Polymer-based protein engineering methods to rationally tune enzyme activity, pH-dependence and stability

ABSTRACT

Using a novel water-soluble, active ester amide-containing functionalized controlled radical polymerization initiator, stimuli responsive polymers have been grown from the surface of a protein, exemplified by chymotrypsin or any protein having surface amino acids that will covalently bind to the active ester amide-containing functionalized initiator. It is shown that changes in temperature or pH can change the conformation of the polymer surrounding the enzyme, which in turn enabled the rational tailoring of enzyme activity and stability. This method has afforded an increase in the activity and stability of the enzyme by an order of magnitude at pH&#39;s where the enzyme is usually inactive or unstable. Multimodal temperature responsive protein-block copolymer conjugates are described.

PRIORITY

This application claims the benefit of U.S. Provisional Application Ser.No. 61/854,321 filed Apr. 22, 2013 and U.S. Provisional Application Ser.No. 61/961,098 filed Oct. 3, 2013, of which the contents of both areincorporated by reference in their entirety.

BACKGROUND

The covalent attachment of polymers to therapeutic proteins (such as theaddition of poly(ethyleneglycol) (PEG) to interferon) has led toextensive commercial use. Since the advent of protein PEGylation in 1979(Veronese, F. M. Biomaterials 2001, 22, 405), much of the researchdevoted to improving the efficacy of protein therapeutics has beenfocused on increasing circulation time and reducing immunogenicity. Asof 2011, at least nine PEGylated protein drugs had been approved by theU.S. Food and Drug Administration (FDA) for treatment of diseasesincluding Hepatitis C (O'Sullivan, A. K.; Buti, M.; Delong, K.; Prasad,M.; Sabater, F. J.; Esteban, R.; Weinstein, M. C. Value Health 2008, 11,A437), acute lymphoblastic leukemia (Dinndorf, P. A.; Gootenberg, J.;Cohen, M. H.; Keegan, P.; Pazdur, R. Oncologist 2007, 12, 991.), andCrohn's disease (Nesbitt, A.; Fossati, G.; Bergin, M.; Stephens, P.;Stephens, S.; Foulkes, R.; Brown, D.; Robinson, M.; Bourne, T. InflammBowel Dis 2007, 13, 1323; Alconcel, S. N. S.; Baas, A. S.; Maynard, H.D. Polym Chem-Uk 2011, 2, 1442.). While PEGylation is a useful tool tohide protein based therapeutics from the immune system, and to increasesize to slow elimination from the body, little additional specificfunctionality is added by PEGylation. In recent years, targeted drugdelivery and drug carriers with responsive functionality (Chilkoti, A.;Dreher, M. R.; Meyer, D. E.; Raucher, D. Adv Drug Deliver Rev 2002, 54,613; Su, J.; Chen, F.; Cryns, V. L.; Messersmith, P. B. J Am Chem Soc2011, 133, 11850; Nasongkla, N.; Bey, E.; Ren, J.; Ai, H.; Khemtong, C.;Guthi, J. S.; Chin, S.-F.; Sherry, A. D.; Boothman, D. A.; Gao, J. NanoLett 2006, 6, 2427) have been investigated to improve efficacy ofcurrent therapeutics.

Polymer conjugation to proteins can be completed using one of twomethods: “grafting to” or “grafting from.” In “grafting to,”pre-synthesized, end functionalized polymers are coupled to accessibleamino acid side chains or end termini on the protein surface. The“grafting-to” technique dominates the literature. The grafting site of afunctionalized synthetic polymer to a protein surface through a couplingreaction is often a random process in which the density and site(s) ofthe grafted polymer cannot be controlled. Naturally, once a firstpolymer chain has “grafting-to” the protein surface steric hindrancewill often prohibit further polymer binding to near-by sites on theprotein surface, resulting in a low density of the grafting polymer.(Lele, B. S.; Murata, H.; Matyjaszewski, K.; Russell, A. J.Biomacromolecules 2005, 6, 3380-3387; Yang, Z.; Domach, M.; Auger, R.;Yang, F. X.; Russell, A. J. Enzyme Microb. Technol. 1996, 18, 82-89.)Although “Grafting to” techniques provide a wide range of polymerizationreactions and monomers to select from, a large excess of polymer isoften required to overcome steric limitations caused by coupledpolymers. In addition, separation of protein-polymer conjugates fromunreacted polymer can prove to be difficult when using the “grafting to”method.

Many pH-responsive polymers show a reversible phase transition betweenexpanded and collapsed forms due to ionization and deionization of theside groups on the polymer that leads to alteration in hydrodynamicvolume and solubility in aqueous media. Early studies on pH-responsivepolymer-protein conjugates showed that conjugation of carboxylatedpolymers such as poly(acrylic acid) to proteins influenced the pHdependence of solubility and activity. (see Charles, M.; Coughlin, R.W.; Hasselberger, F. X. Biotech. Bioeng. 1974, 16, 1553-1556; VanLeemputten, E.; Horisberger, M. Biotech. Bioeng. 1976, 18, 587-590)Thermo-responsive polymers respond to changes in temperature and exhibitreversible transitions between collapsed and expanded forms attemperatures above and below their critical solution temperature. Forexample, poly(N-isopropylacrylamide) (pNIPAm) has a low criticalsolution temperature (LCST) around 32° C. in the aqueous solution. Attemperatures above the LCST, pNIPAm becomes dehydrated and collapsesinto micelle-like particles which precipitate from solution. Thisproperty of temperature-responsive polymer-protein conjugates has beenused to enhance purifications using pNIPAm-modified Protein A andmonoclonal antibodies that were synthesized with the “grafting-to”approach. “Grafted-to” enzyme-pNIPAm conjugates are unpredictablehowever in that some enzymes exhibit modulated bioactivity but others donot.

In order to provide an alternative approach to synthesis ofpolymer-enzyme conjugates that would allow higher densities, and finersite control, a protein surface initiated “grafting from” technique waspreviously developed (Lele et al, Biomacromolecules 2005, 6, 3380-3387)This resulted in a higher density of polymer on the enzyme surface butbecause the initiator binding and polymerization were done in an organicsolvent-water biphasic medium the recovery of activity was low and thedensity was still not optimal. (see also Heredia, K. L.; Bontempo, D.;Ly, T.; Byers, J. T.; Halstenberg, S.; Maynard, H. D. J Am Chem Soc2005, 127, 16955)

“Grafting from” techniques initiate polymerization directly from thesurface of proteins using controlled radical polymerization. Most often,either atom transfer radical polymerization (ATRP) (see Lele, B. S.;Murata, H.; Matyjaszewski, K.; Russell, A. J. Biomacromolecules 2005, 6,3380; Nicolas, J.; San Miguel, V.; Mantovani, G.; Haddleton, D. M. Chem.Commun. 2006, 4697; Heredia, K. L.; Bontempo, D.; Ly, T.; Byers, J. T.;Halstenberg, S.; Maynard, H. D. J. Am. Chem. Soc. 2005, 127, 16955; Qi,Y.; Amiram, M.; Gao, W.; McCafferty, D. G.; Chilkoti, A. Macromol. RapidCommun. 2013, 34, 1256; Gao, W.; Liu, W.; Mackay, J. A.; Zalutsky, M.R.; Toone, E. J.; Chilkoti, A. Proc. Natl. Acad. Sci. U.S.A. 2009, 106,15231) or reversible-addition fragmentation chain transfer (RAFT) (Liu,J.; Bulmus, V.; Herlambang, D. L.; Barner-Kowollik, C.; Stenzel, M. H.;Davis, T. P. Angew. Chem., Int. Ed. 2007, 46, 3099; De, P.; Li, M.;Gondi, S. R.; Sumerlin, B. S. J. Am. Chem. Soc. 2008, 130, 11288) areused, because each provide low polydispersity indices (PDI), a largelibrary of monomers, and biologically relevant reaction conditions(aqueous solvent and ambient temperature). In “grafting from,” unreactedmonomer is easily separated from the bioconjugate and high polymerdensity is achieved more easily due to the lack of steric limitationsseen in “grafting to.” One drawback to “grafting from” is the necessityto have vinyl monomers for radical polymerization. Thus, some polymers,such as PEG, must be slightly modified to use with the “grafting from”approach.

Another major limitation of the current “grafting-from” ATRP techniquesfor polymer based protein engineering is in the attachment orimmobilization of an ATRP-initiator to the enzyme. Until recently mostfunctionalized ATRP initiator compounds were insoluble or of lowsolubility in aqueous solution. Thus, immobilization of an ATRPinitiator to a protein was performed in mixtures of water and organicsolvents such as dichloromethane, methanol, DMF, or DMSO. (Nicolas, J.;San Miguel, V.; Mantovani, G.; Haddleton, D. M. Chem. Commun. 2006, 46,4697-4699; Magnusson, J. P.; Bersani, S.; Salmaso, S.; Alexander, C.;Caliceti, P. Bioconjugate Chem. 2010, 21, 671-678; Ya

ayan, G.; Saeed, A. O.; Fernández-Trillo, F.; Allen, S.; Davies, M. C.;Jangher, A.; Paul, A.; Thurecht, K. J.; King, S. M.; Schweins, R.;Griffiths, P. C.; Magnusson, J. P.; Alexander, C. Polym. Chem. 2011, 2,1567-1578; Averick, S.; Simakova, A.; Park, S.; Konkolewicz, D.;Magenau, A. J. D.; Mehl, R. A.; Matyjaszewski, K. ACS Macro Lett. 2011,1, 6-10.) Such mixtures often lead to inactivation and/or denaturationof enzymes during the immobilization reaction. Further, initiatorimmobilizations performed on chymotrypsin (CT) in a biphasic solutionand on trypsin in 2% DMSO resulted in 21-50% and 46% occupation ofavailable conjugation sites, respectively.

Techniques to synthesize protein-polymer conjugates have developedrapidly in recent years due to advancements in both protein and polymerscience. One of the first, and still most common polymers to attach toproteins is poly(ethylene glycol) (PEG), (see Alconcel, S. N. S.; Baas,A. S.; Maynard, H. D. Polym. Chem. 2011, 2, 1442), which imparts stealthproperties on the protein by reducing immunogenicity and increases invivo stability by slowing renal clearance and degradation. However, thispolymer does not add specific functionality to the protein and oftenresults in reduced activity. (see Veronese, F. M. Biomaterials 2001, 22,405) More recently, different polymers have been utilized to synthesize“smart conjugates” (see Hoffman, A. S.; Stayton, P. S. Prog. Polym. Sci.2007, 32, 922 that respond to external stimuli such as pH (Lackey, C.A.; Murthy, N.; Press, O. W.; Tirrell, D. A.; Hoffman, A. S.; Stayton,P. S. Bioconjugate Chem. 1999, 10, 401; Strozyk, M. S.; Chanana, M.;Pastoriza-Santos, I.; Pérez-Juste, J.; Liz-Marzán, L. M. Adv. Funct.Mater. 2012, 22, 1436.). In addition, specific polymer choices fortailored applications, such as increased substrate affinity (Keefe, A.J.; Jiang, S. Y. Nat. Chem. 2012, 4, 60) have been reported.Polymer-based protein engineering refers to these tailored polymerconjugation applications that target problems that previously could onlypotentially be solved with molecular biology-dependent techniques.

Poly(sulfobetaine methacrylamide) (pSBAm) andpoly(N-isopropylacrylamide) (pNIPAm) are two polymers that have beeninvestigated for a wide range of chemical and biological applications.Specifically, pNIPAm can be used in applications for cardiac repair(Naito, H.; Takewa, Y.; Mizuno, T.; Ohya, S.; Nakayama, Y.; Tatsumi, E.;Kitamura, S.; Takano, H.; Taniguchi, S.; Taenaka, Y. ASAIO J. 2004, 50,344), protein drug release, and biomolecule separations (Zhou, P.; Yu,S. B.; Liu, Z. H.; Hu, J. M.; Deng, Y. Z. J. Chromatogr. A 2005, 1083,173). pSBAm is used frequently for non-fouling surface modification(Zhang, Z.; Finlay, J. A.; Wang, L.; Gao, Y.; Callow, J. A.; Callow, M.E.; Jiang, S. Langmuir 2009, 25, 13516; Smith, R. S.; Zhang, Z.;Bouchard, M.; Li, J.; Lapp, H. S.; Brotske, G. R.; Lucchino, D. L.;Weaver, D.; Roth, L. A.; Coury, A.; Biggerstaff, J.; Sukavaneshvar, S.;Langer, R.; Loose, C. Sci. Transl. Med. 2012, 4, 153ra132). Both pSBAmand pNIPAm respond to changes in temperature by predictable alterationsin polymer folding. pNIPAm has a lower critical solution temperature(LCST), where above ˜32° C. in deionized water the polymer experiences areversible collapse, in which it becomes hydrophobic and dehydrated.(Schild, H. G. Prog. Polym. Sci. 1992, 17, 163.) pSBAm exhibits asimilar, but opposite behavior known as upper critical solutiontemperature (UCST) phase transition. pSBAm UCST values are moredependent on molecular weight than the LCST of pNIPAm, but below a giventemperature polymer chains collapse from a coil to globule orientationas they phase separate and become insoluble in aqueous media. (Chen, L.;Honma, Y.; Mizutani, T.; Liaw, D. J.; Gong, J. P.; Osada, Y. Polymer2000, 41, 141.) Free block copolymers with both UCST and LCST propertieshave been reported previously (Arotcarena, M.; Heise, B.; Ishaya, S.;Laschewsky, A. J. Am. Chem. Soc. 2002, 124, 3787; Weaver, J. V. M.;Armes, S. P.; Butun, V. Chem. Commun. 2002, 2122), but protein-polymerconjugates are most often only synthesized with single temperatureresponsiveness imparted by homopolymer conjugation (Kulkarni, S.;Schilli, C.; Muller, A. H. E.; Hoffman, A. S.; Stayton, P. S.Bioconjugate Chem. 2004, 15, 747; Boyer, C.; Bulmus, V.; Liu, J. Q.;Davis, T. P.; Stenzel, M. H.; Barner-Kowollik, C. J. Am. Chem. Soc.2007, 129, 7145). While block copolymers are sometimes conjugated toproteins with the “grafting to” approach, there are few reports of blockcopolymers being grown from proteins using “grafting from.” Previously,Sumerlin and coworkers used “grafting from” to synthesize a blockcopolymer using two consecutive RAFT polymerizations from lysozyme (Li,H. M.; Li, M.; Yu, X.; Bapat, A. P.; Sumerlin, B. S. Polym. Chem. 2011,2, 1531) and bovine serum albumin (Li, M.; Li, H. M.; De, P.; Sumerlin,B. S. Macromol. Rapid Commun. 2011, 32, 354.). Kulkarni et al.synthesized a block copolymer with modified temperature sensitivity, butused the “grafting to” process for protein conjugation (see Kulkarni,S.; Schilli, C.; Grin, B.; Muller, A. H. E.; Hoffman, A. S.; Stayton, P.S. Biomacromolecules 2006, 7, 2736).

SUMMARY OF THE INVENTION

The inventors hypothesized that if one could find a way to growresponsive polymers from the surface of a protein, for example, anenzyme, then one could influence the functionality of catalyticallyactive proteins in a controlled reliable manner by polymer-based proteinengineering (PBPE) as an alternative to site-directed mutagenesis.

In the “grafting-from” approach using the methods and macroinitiatordescribed hereinto, a wide range of monomers can be used for conjugationand the molecular weight of the grafted polymer on the conjugate can beexquisitely controlled with a narrow polymer distribution. Thus,“grafting-from” bioconjugates can be rationally designed using themethods described herein, allowing structure-function relationshipsbetween the polymer and the protein to predict the functionality of theresulting covalent conjugate. One important variable that had heretoforebeen unresolved was the control of polymer density.

The various embodiments of the methods of the present invention permitthe growth of polymer chains from the surface of proteins. In variousembodiments, the polymer chains may be stimuli responsive polymers, suchas pH- and temperature-responsive polymers, grown from the surface ofproteins in order to tune the pH- and temperature-dependence ofbioactivity. Controlled manipulation of the bioactivity of proteins, andin particular, enzymes, opens the door to a new class of biomoleculesthat may be used, for example, in therapeutic applications.

An embodiment of the composition may comprise a densely modifiedprotein-polymer conjugate with a density of polymer chains per unitsurface area exceeding one polymer chain per 20 nm² of protein surfacearea. The density may be greater than one polymer chain per 10 nm² ofprotein surface area or in some embodiments, densities between about onepolymer chain per less than 20 nm² of protein surface area.

In this invention, a novel, water soluble, active ester-functionalizedamide-containing controlled radical polymerization (CRP) initiator isdesigned and immobilized to the amines on the surface of a protein inaqueous solution. The CRP initiator comprises the general structure

wherein

X is a halogen, such as Br, Cl, or F, or a chain transfer agent;

R₁ is H or alkyl;

R₂ is an active ester moiety; and

n is an integer from 1 to 6.

The active ester moiety may be N-oxysuccinimde ester, nitrophenyl ester,pentahalophenyl ester wherein the halogen is F or Cl, 1-oxybenzotriazoleester, or 2-oxy-4,6-dimethyloxy-1,3,5-triazine ester. The chain transferagent may be any suitable known chain transfer agent used in a RAFTpolymerization procedure. See, for example, Handbook of RadicalPolymerization, K. Matyjaszewski and T. Davis, Ed., John Wiley & Sons,Inc. pub. (2002), Section 12.4, incorporated herein by reference.Exemplary chain transfer agents of the general structure

fall into four classes of thiocarbonylthio agents: (1) dithioesters,where Z is aryl or alkyl, (2) trithiocarbonates where Z is a substitutedsulphur, (3) dithiocarbonates (xanthates), where Z is substitutedoxygen, and (4) dithiocarbamates, where Z is substituted nitrogen.

In one example, the active ester-functionalized amide-containing CRPinitiator is an N-2-bromo-2-methylpropanoyl-β-alanine N′-oxysuccinimideester.

In various embodiments, the active ester-functionalized amide-containingCRP initiator is an NHS-functionalized ATRP initiator which may beimmobilized to the surface reactive amino acid side chains of a protein,for example, an enzyme. In one embodiment, the activeester-functionalized amide-containing controlled radical polymerization(CRP) initiator may be immobilized to lysines on a serine proteaseα-chymotrypsin (CT) surface in aqueous solution. Herein, it isdemonstrated that in various embodiments, the initiator occupied aplurality of the binding sites on the surface of the protein, and moreparticularly, a majority of binding sites, and of significance, occupiedfrom about 85-100%, and in some embodiments, occupied all of the bindingsites, or about 90% of the binding sites, or about 86% of the bindingsites, and at least 85% of the binding sites on the surface of theenzyme, without significantly affecting enzyme activity.

In various embodiments, the method of the invention comprisesimmobilizing an active ester-functionalized amide-containing CRPinitiator in an aqueous solution on each of a majority of amino bindingsites on a protein surface to form a protein-initiator conjugate,isolating the protein-initiator conjugate, mixing a first group ofmonomers having one or more desired properties with theprotein-initiator conjugate, polymerizing the monomers from theprotein-initiator conjugate to grow a polymer under controlled radicalpolymerization conditions to form a protein-polymer conjugate; and,isolating the protein-polymer conjugate. The chain length of thepolymers is controlled by adjusting the molar concentration of the firstgroup of monomers added to the protein initiator conjugate to a desiredamount.

The controlled radical polymerization conditions may include ATRP orRAFT polymerization procedures. If ATRP is the polymerization procedureof choice, then the functional group, X, in the activeester-functionalized amide-containing CRP initiator structure (I) aboveis preferably a halogen. If RAFT is the polymerization procedure ofchoice, then the functional group, X, in the initiator structure (I)above is a chain transfer agent.

The step of immobilizing the initiator may include mixing protein andthe active ester-functionalized amide-containing CRP initiator inbuffer, for example, at a pH of about 8 to 9, and stirring for a periodof time sufficient to allow the formation of covalent bonds between theinitiator and the plurality of amino binding sites. The step ofisolating the protein-initiator conjugate may include removing unreactedand unattached compounds from the solution, by for example, passing thesolution through a dialysis membrane. Similarly, the step of isolatingthe protein-polymer conjugate may be done by passing the mixture througha dialysis membrane under refrigeration for a period of time sufficientto remove catalyst and unreacted monomer. The method may furthercomprise the step of lyophilizing the protein-polymer conjugate.

The step of mixing the monomers with the protein-initiator conjugateunder controlled radical polymerization conditions may include mixingunder ATRP conditions, for example, mixing in buffer and removing oxygenfrom the mixture, adding a deoxygenated ligand to a separate aqueouscopper catalyst solution, transferring the copper-ligand catalystsolution to the protein-initiator conjugate and monomer mixture, andstirring at 4-25° C. for a sufficient time to allow polymerization toproceed. Removing oxygen from the protein-initiator conjugate andmonomer mixture may be done by bubbling Ar or N₂ through the mixture.

The protein may be an enzyme selected from the group consisting ofchymotrypsin, lysozyme, β-Galactosidase, carbonic anhydrase, glucoseoxidase, laccase, and acetylcholinesterase. Other proteins and enzymesor hormones may be used provided they have surface amino groups, such assurface lysine or cysteine groups available for covalent binding withthe active ester of the active ester-functionalized amide-containing CRPinitiator. Suitable surface amino groups include any amino acid residuehaving surface reactive amino acids that can covalently bind to theactive ester of the amide-containing initiator. The structures of aminoacids are well known. Those skilled in the art may determine suitableamino acid residues that can covalently bind to the active ester byreference to the literature.

The polymers used for the protein-polymer conjugate are preferablystimuli responsive polymers that respond to at least one stimulus. Thestimulus may be one or both of pH and temperature. The polymer may alsobe one that changes the charge of the protein. The protein-polymerconjugate may be, for example, a chymotrypsin modified through highdensity attachment of thermo-responsive polymers.

In certain embodiments of the method described herein, theprotein-polymer conjugate formed from the controlled radicalpolymerization may be a protein-homopolymer conjugate of differentpolymer chain lengths. Following the polymerization of the first groupof monomers from the protein-initiator conjugate, the method may furtherinclude the steps of mixing a second group of monomers having one ormore desirable properties with the protein-homopolymer conjugate undercontrolled radical polymerization conditions to form a block copolymer.In an exemplary embodiment, the block copolymer may be a dualtemperature responsive enzyme-pSBAm-block-pNIPAm conjugate havingdifferent polymer chain lengths and molecular weights. In anotherexemplary embodiment, the protein may be, for example, an enzyme with asurface charge modified by growing cationic pQA from multiple sites onthe surface of enzyme. In another exemplary embodiment, the polymersgrown from the plurality of surface amino sites of the protein core mayform a high density cationic polymer shell around the protein core.

The method described herein provides a macroinitiator comprising a watersoluble active ester-functionalized amide-containing controlled radicalpolymerization initiator having the structure

wherein X is a halogen, such as Br, F, or Cl, or a chain transfer agent;R₁ is H or alkyl; R₂ is an active ester moiety; and n is an integer from1 to 6, covalently bound to each of a plurality of surface aminoresidues on a protein, such as lysine or cysteine residues, and in someembodiments, the N-terminal amino residue. The activeester-functionalized amide-containing CRP initiator may bind to at least85% of the surface amino residues on the protein, and may alsocovalently bind to the N-terminus of the protein. Examples of the activeester moieties that may be used in the initiator are described above.

An embodiment of the method described herein provides a protein-polymerconjugate that comprises an enzyme core having surface amino residuescovalently bound at each of at least a majority of the surface aminoresidues, and preferably at least 85% or more of the surface aminoresidues, to a stimuli responsive polymer grown from the surface of theenzyme under controlled radical polymerization conditions, such as ATRPor RAFT. Exemplary enzyme may be selected from the group consisting ofchymotrypsin, lysozyme, β-Galactosidase, carbonic anhydrase, glucoseoxidase, laccase, and acetylcholinesterase. Those skilled in the artwill recognize that other proteins, for example, other enzymes, withfree reactive amino acid side chains residues on one or both of thesurface or the N-terminus, may be used in the method described herein.The polymer of the protein-polymer conjugate may form a block copolymer,or may form a cationic shell around the enzyme core.

In certain embodiments, the “grafting from” synthesis of a blockcopolymer may include carrying out two consecutive ATRP reactions fromthe surface of a protein. For example, temperature sensitive polymers,such as pSBAm and pNIPAm, may be grafted from the surface ofchymotrypsin (CT) using two consecutive ATRP reactions.

Using polymer-based protein engineering (PBPE) with aqueous atomtransfer radical polymerization (ATRP), three different molecular weightCT-pSBAm-block-pNIPAm bioconjugates were synthesized that respondedstructurally to both low and high temperature. In the block copolymergrown from the surface of the enzyme, upper critical solutiontemperature (UCST) phase transition was dependent on the chain length ofthe polymers in the conjugates, whereas lower critical solutiontemperature (LCST) phase transition was independent of molecular weight.Each CT-pSBAm-block-pNIPAm conjugate showed temperature dependentchanges in substrate affinity and productivity when assayed from 0 to40° C. In addition, these conjugates showed higher stability to harshconditions, including temperature, low pH, and protease degradation.Indeed, the PBPE-modified enzyme was active for over eight hours in thepresence of a stomach protease at pH 1.0. Using PBPE a dual zone shellsurrounding each molecule of enzyme was created. The thickness of eachzone of the shell was engineered to be separately responsive totemperature.

Atom transfer radical polymerization-based protein engineering of, forexample, the enzyme chymotrypsin with a cationic polymer, such aspoly(quaternary ammonium) may be used to tune the activity, stability,and inhibitor binding of the enzyme. Poly(quaternary ammonium), forexample, was grown from the surface of the enzyme using atom transferradical polymerization after covalent attachment of a protein reactive,water soluble NHS-amide-containing halo functionalized initiator,sometimes referred to herein for brevity as an ATRP initiator. This“grafting from” conjugation approach generated highly concentratedcationic ammonium ions around the biocatalytic core. After modification,bioactivity was increased at low pH relative to the activity of thenative enzyme. In addition, substrate affinity was increased afterconjugation over a wide range of pH's. The massively cationicchymotrypsin was also more stable at extremes of temperature and pH.Most interestingly, the methods allow rational control of the binding oftwo oppositely charged protease inhibitors, aprotinin and Bowman-Birktrypsin-chymotrypsin inhibitor from glycine max, to the cationicderivative of chymotrypsin.

Poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) exhibits a phasetransfer between super-hydrophilic and hydrophobic characteristics belowand above its pK_(a). The chains of PDMAEMA are expanded in aqueoussolution when tertiary amine groups of PDMAEMA are protonated andhydrated below the pK_(a). In contrast, the polymer chains are collapsedby deprotonation and dehydration of the amine group above the pK_(a).There are also conformational changes in PDMAEMA below and above its LowCritical Solution Temperature (LCST). PDMAMEA is used herein as a modelto determine how “grafted-from” stimuli-responsive protein-polymerconjugates can be controlled with environmental variables such as pH,temperature and solvent. Growing the polymer from the surface of CT, itwas demonstrated by several examples that changes in temperature or pHcan change predictably the conformation of the polymer surrounding theenzyme, which in turn enabled the rational tailoring of enzyme activityand stability. Using this method and approach, the activity andstability of CT has increased by an order of magnitude at pH's where theenzyme is usually inactive or unstable.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates an overview of Polymer-based protein engineering(PBPE) using ATRP with DMAEMA and chymotrypsin.

FIG. 2 illustrates the ¹H NMR and MALDI TOF Mass spectra of native CTand CT-ATRP initiator conjugate. Spectrum a is ¹H NMR of CT-ATRPinitiator conjugate in D₂O and spectrum b is of native CT in D₂O.Spectrum c is MALDI-TOF-MS of native CT, and d is that of the CT-ATRPinitiator conjugate.

FIG. 3 illustrates the gel permeation chromatography (GPC) experimentsfor molecular weight determination of cleaved PDMAEMA from theconjugates.

FIG. 4 illustrates the hydrodynamic diameter of native andpolymer-modified chymotrypsin as a function of pH. a) pH-dependence ofthe hydrodynamic diameter of native CT and conjugates; b) Hydrodynamicdiameter (for native and modified enzyme) relative to those at pH 5; c)schematic representation of the impact of pH on the conformation of thegrafted PDMAEMA chains below and above pH 8.

FIG. 5 illustrates the impact of Polymer-Based Protein Engineering onthe relative Michaelis-Menten kinetics of the chymotrypsin-catalyzedhydrolysis of Suc-AAPF-pNA. (a) pH-dependence of turnover numbers(k_(cat)) for PDMAEMA-CT conjugates relative to those for the nativeenzyme. The inset schematic illustrates how protonated PDMAEMA couldstabilize the deprotonation of Asp 102 below pH 8; (b) pH-dependence ofsubstrate affinities (K_(M)) of chymotrypsin conjugates relative tothose for the native enzyme. The inset schematic illustrates how thedeprotonated grafted PDMAEMA could sterically hinder the active site ofCT above pH 9; (c) pH dependence of catalytic efficiency (k_(cat)/K_(M))of CT conjugates relative to the native enzyme.

FIG. 6 shows the GPC traces of free PDMEMA prepared separately.

FIG. 7 illustrates the temperature dependence of kinetic constants fornative and PBPE-modified chymotrypsin. (a) temperature dependenceactivity; (b) temperature dependence of specificity; (c) temperaturedependence of productivity.

FIG. 8 illustrates the pH-dependence of the rate of irreversibleinactivation of and native PBPE-modified chymotrypsin at 40° C. Nativechymotrypsin and the conjugates were incubated at pH 7.0 (a), pH 8.0 (b)and pH 9.0 (c). All enzyme assays were performed at 25° C. The insetschematics illustrate the likely conformation of the grafted PDMAEMAchains at each pH (not to scale).

FIG. 9 illustrates the low critical solution temperature of theCT-PDMAEMA example conjugates.

FIG. 10 shows cloud point curves for CT-pSBAm-block-pNIPAm conjugates(CT-35/39-open diamond, CT-50/67-closed square, CT-90/100-closedcircle). Each conjugate (3 mg/mL) was incubated in 0.1 M sodiumphosphate buffer (pH=8.0), heated/cooled at ±0.5° C./min, and absorbanceat 490 nm was recorded.

FIGS. 11A-C illustrate Temperature dependence of enzyme (A) specificity(K_(M)), (B) activity (k_(cat)), and (C) productivity (k_(cat)/K_(M))relative values in the hydrolysis of NS-AAPF-pNA byCT-pSBAm-block-pNIPAm conjugates (CT-35/39-open diamond/dash-dot line,CT-50/67-closed circle/large dash line, CT-90/100-closed square/shortdash line) relative to native CT in 0.1 M sodium phosphate buffer(pH=8.0). Values for native chymotrypsin at increasing temperature (2.5°C., 7° C., 16.5° C., 24.5° C., 33° C., and 37.5° C.) are as follows:K_(M)(μM)-30±5.1, 29±6.9, 37±8.6, 51±8.7, 57±5.0, 59±6.9;k_(cat)(sec⁻¹)-8.9±0.4, 9.7±0.5, 16±0.9, 25±1.3, 36±1.3, 47±1.7;k_(cat)/K_(M)(sec⁻¹/μM)-0.33±0.06, 0.34±0.09, 0.43±0.10, 0.50±0.1,0.80±0.1. CT-pSBAm-block-pNIPAm kinetic values are shown in SI Table 1.

FIG. 12 is a schematic of the hypothesized effect of pSBAm and pNIPAmpolymer collapse on substrate affinity (K_(M)). At 25° C., both pSBAmand pNIPAm were in their extended conformation and allowed Suc-AAPF-pNAaccess to CT active site. At temperatures below pSBAm UCST and abovepNIPAm LCST, polymer collapse inhibited access to the active site forSuc-AAPF-pNA due to steric blocking. At temperatures below pSBAm UCST,this effect is hypothesized to be more pronounced than at temperaturesabove pNIPAm LCST, because the pSBAm block was closer to the enzyme corethan the pNIPAm block.

FIGS. 13A-C provide graphs showing the rate of irreversible inactivationfor CT-pSBAm-block-pNIPAm conjugates (CT-35/39-open diamonds,CT-50/67-closed squares, and CT-90/100-closed circles), native CT (opencircles), and native CT with pSBAm-block-pNIPAm in solution (opensquares) at 37° C. in (a) 0.1 M sodium phosphate buffer (pH=8.0), (b)167 mM HCl (pH=1), and (c) 167 mM HCl with 19 nM pepsin. Residualactivity was calculated as the activity remaining from t=0. All assayswere conducted at 25° C.

FIG. 14 illustrates the dependence of native and CT conjugatehydrodynamic diameter during incubation in 167 mM HCl (pH 1) at 37° C.Size values are presented as ratios of each samples size at time zero.Increased hydrodynamic diameter (D_(h)) indicates protein unfolding.D_(h) values at time zero were: native CT (open circle)-6.3±0.5 nm,CT-35/39 (open diamond)-51±9.7 nm, CT-50/67 (closed circle)-63±14 nm,CT-90/100 (closed square)-72±12 nm.

FIG. 15 shows the impact of polymer-based protein engineering on therelative Michaelis-Menten kinetics of chymotrypsin catalyzed hydrolysisof Suc-AAPF-pNA. A. pH dependence of relative turnover number forchymotrypsin-pQA conjugates relative to native chymotrypsin. (CT-pQA27;open square, CT-pQA54; open triangle, CT-pQA108; open diamond,CT-pQA198; open circle) B. pH dependence of relative substrate affinityof chymotrypsin conjugates C. pH dependence of catalytic efficiency ofchymotrypsin conjugates.

FIG. 16 shows the temperature dependence of irreversible inactivation ofchymotrypsin and chymotrypsin-pQA. (A) 50° C. (B) 60° C. Conjugates(CT-pQA₂₇; open square, CT-pQA₅₄; open triangle, CT-pQA₁₀₈; opendiamond, CT-pQA₁₉₈; open circle) and native chymotrypsin (closed circle)were incubated in 100 mM sodium phosphate buffer (pH 8) at 3.9 μM for 8hours.

FIG. 17 is a graph showing the rate of irreversible inactivation in 167mM HCl aq. (pH 1.0) at 37° C. for native chymotrypsin and chymotrypsinconjugates (CT-pQA₂₇; open square, CT-pQA₅₄; open triangle, CT-pQA₁₀₈;open diamond, CT-pQA₁₉₈; open circle) and native CT (closed circle).

FIG. 18 shows the impact of polymer-based protein engineering on theinhibitor binding to chymotrypsin-pQA conjugates. Concentrationdependence of aprotinin (AP) (A) and Bowman-Birk trypsin-chymotrypsininhibitor from glycine max (GM) (B) on the relative enzymatic activityof conjugates (CT-pQA₂₇; open square, CT-pQA₅₄; open triangle,CT-pQA₁₀₈; open diamond, CT-pQA₁₉₈; open circle) and native CT (closedcircle). The inset picture shows the hypothesized effect ofelectrostatic attraction and repulsion on inhibitor binding.

FIG. 19 shows the NMR spectra of chymotrypsin-pQA conjugates usingCT-pQA₅₀ dissolved in D₂O (10 mg/mL). The NMR spectra peaks identifyingcorrelating proton shifts are specified above.

FIG. 20 shows the NMR spectra of pQA polymers cleaved from the surfaceof chymotrypsin using acid hydrolysis were determined using pQA₂₀₀ (10mg/mL) in D₂O. Chymotrypsin-pQA conjugates were dissolved in 6 N HCl(10-20 mg/mL), followed by three freeze-pump-thaw cycles to remove airfrom the mixture. Conjugates were incubated at 110° C. for 24 hr.Dialysis filtering (MwCO 1000 Da) against DI water was used to removedigested enzyme and HCl from the solution.

FIG. 21 represents the results of size exclusion chromatography (SEC)used to determine the molecular weight of pQA polymers cleaved from thesurface of chymotrypsin using acid hydrolysis. Each of the four pQAconjugates was dissolved at 5 mg/mL using 0.1 M sodium phosphate buffer(pH 2) with 0.2 vol % TFA as the eluent. Samples were run at a flow rateof 1 mL/min. Poly(ethylene glycol) standards were used to determine themolecular weight.

FIGS. 22A and B show (A) Apparent K_(M) and V_(max) values weredetermined for native CT incubated with aprotinin protein inhibitor bymonitoring enzyme catalyzed hydrolysis of Suc-AAPF-pNA after mixinginhibitor (0-0.49 μM), enzyme (39 nM), and substrate (0-750 μM) togetherat the same time. Apparent K_(M) and V_(max) values were calculatedusing EnzFitter by Michelis-Menten curve fitting of substrate againstinitial velocity plots. Values are shown in Table 11. (B) Secondaryplots with calculated apparent K_(M) and V_(max) values were used todetermine inhibition constants of aprotinin towards native chymotrypsin.

FIGS. 23A and B show (A) Substrate/initial velocity plots and (B)secondary plots to determine inhibitor constant for chymotrypsin-pQA₂₀₀with aprotinin in 0.1 M sodium phosphate buffer (pH 8) at 25° C. Theprocedure used was the same as described above for aprotinin and nativechymotrypsin.

FIGS. 24A and B show (A) Substrate/initial velocity plots and (B)secondary plots to determine inhibitor constant for native chymotrypsinwith GM in 0.1 M sodium phosphate buffer (pH 8) at 25° C. The procedureused was the same as described above for aprotinin and nativechymotrypsin except less inhibitor was used (0-0.294 μM).

FIGS. 25A and B show (A) Substrate/initial velocity plots and (B)secondary plots to determine inhibitor constant for chyomtrypsin-pQA₂₀₀with GM in 0.1 M sodium phosphate buffer (pH 8) at 25° C. The procedureused was the same as described above for aprotinin and nativechymotrypsin except less inhibitor was used (0-0.294 μM).

FIG. 26 is a Table for Experimental Section II showing the temperaturedependence of chymotrypsin and bioconjugate activity, specificity andproductivity for the hydrolysis of Suc-AAPF-pNA.

FIG. 27 shows the MALDI-TOF-MS spectra for native chymotrypsin (top) andATRP initiator modified chymotrypsin (bottom), described in ExperimentalSection IV.

FIG. 28 shows UCST cloud point curves for CT-pSBAm conjugates describedin Experimental Section IV.

FIGS. 29-31 show NMR spectra for pSBAm₃₅-block-pNIPAm₃₉ in D₂O,pSBAm₅₀-block-pNIPAm₆₇ in D₂O, and pSBAm₉₀-block-pNIPAm₁₀₀ in D₂O, eachcleaved from chymotrypsin using acid hydrolysis.

DETAILED DESCRIPTION AND BEST MODE OF IMPLEMENTATION

The attachment of synthetic polymers to proteins influences activity andstability and offers a means to add novel functions to the protein in avariety of microenvironments. The methods and compositions describedherein add significantly to the body of work in this field by allowing acontrolled rational approach to the attachment method that permitsmodification of proteins in a controlled reliable manner. By usingmonomers to grow polymers that have been determined to be responsive tostimuli to effect a desired property, the protein of interest can bemodified with confidence that the polymer protein conjugate will exhibitthe desired property, and importantly, will function in the manner forwhich it was designed in a relevant microenvironment.

To provide an overall understanding, certain illustrative embodimentswill now be described; however, it will be understood by one of ordinaryskill in the art that the systems and methods described herein can beadapted and modified to provide systems and methods for other suitableapplications and that other additions and modifications can be madewithout departing from the scope of the systems and methods describedherein.

Unless otherwise specified, the illustrated embodiments can beunderstood as providing exemplary features of varying detail of certainembodiments, and therefore unless otherwise specified, features,components, modules, and/or aspects of the illustrations can becombined, separated, interchanged, and/or rearranged without departingfrom the disclosed systems or methods.

Introduction

For convenience, certain terms employed in the specification, examples,and appended claims are collected here. Unless defined otherwise, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art.

Other than in the examples herein, or unless otherwise expresslyspecified, all of the numerical ranges, amounts, values and percentages,such as those for amounts of materials, elemental contents, times andtemperatures of reaction, ratios of amounts, and others, in thefollowing portion of the specification and attached claims, may be readas if prefaced by the word “about” even though the term “about” may notexpressly appear with the value, amount, or range. Accordingly, unlessindicated to the contrary, the numerical parameters set forth in thefollowing specification and claims are approximations that may varydepending upon the desired properties sought to be obtained by thepresent invention. At the very least, and not as an attempt to limit theapplication of the doctrine of equivalents to the scope of the claims,each numerical parameter should at least be construed in light of thenumber of reported significant digits and by applying ordinary roundingtechniques.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. Any numerical value, however, inherently contains errornecessarily resulting from the standard deviation found in itsunderlying respective testing measurements. Furthermore, when numericalranges are set forth herein, these ranges are inclusive of the recitedrange end points (i.e., end points may be used). When percentages byweight are used herein, the numerical values reported are relative tothe total weight.

Also, it should be understood that any numerical range recited herein isintended to include all sub-ranges subsumed therein. For example, arange of “1 to 10” is intended to include all sub-ranges between (andincluding) the recited minimum value of 1 and the recited maximum valueof 10, that is, having a minimum value equal to or greater than 1 and amaximum value of equal to or less than 10. The articles “a” and “an” areused herein to refer to one or to more than one (i.e., to at least one)of the grammatical object of the article. By way of example, “anelement” means one element or more than one element.

The term “microenvironment” refers to localized conditions within alarger area. For example, association of two molecules within a solutionmay alter the local conditions surrounding the associating moleculeswithout affecting the overall conditions within the solution.

The term “protein”, and the terms “polypeptide” and “peptide” which areused interchangeably herein, refers to a polymer of amino acids.

The term “densely modified” with respect to the protein-polymerconjugates described herein means a density of polymer chains per unitsurface area of protein of one polymer chain per 1 to 10 nm² of proteinsurface area, or a density such that a majority of amine binding sites,and in various embodiments, from about 85-100%, and in some embodiments,all of the amine binding sites, or about 90% of the amine binding sites,or about 86% of the amine binding sites, and at least 85% of the aminebinding sites on the surface of the protein are bound to a polymerchain.

The term “ligand” as used herein with respect to a controlled radicalpolymerization process means a moiety used to solubilize the transitionmetal salt in the reaction media and to adjust the redox potential andhalogenophilicity of the metal center forming a complex with anappropriate reactivity and dynamics for the atom transfer. Exemplaryligands are described in Handbook of Radical Polymerization, K.Matyjaszewski and T. Davis, Ed., John Wiley & Sons, Inc. pub. (2002), pp553-555, 567, incorporated herein by reference.

The terms “active ester functionalized amide-containing controlledradical polymerization initiator”, “controlled radical polymerizationinitiator”, or “CRP initiator”, or “NHS-functionalized amide-containinginitiator” or “NHS-functionalized ATRP initiator”, or “ATRP initiator,”and the like, are used herein interchangeably, and refer to the watersoluble, active ester-functionalized amide-containing controlled radicalpolymerization (CRP) initiator used in the polymer-based proteinengineering methods described herein and comprising the generalstructure

wherein X is a halogen, such as Br, Cl, or F, or a chain transfer agent;R₁ is H or alkyl; R₂ is an active ester moiety; and n is an integer from1 to 6. The active ester moiety may be N-oxysuccinimde ester,nitrophenyl ester, pentahalophenyl ester wherein the halogen is F or Cl,1-oxybenzotriazole ester, or 2-oxy-4,6-dimethyloxy-1,3,5-triazine ester.

Exemplary chain transfer agents that may form X in structure (I) may, incertain embodiments, comprise the general structure

and fall into four classes of thiocarbonylthio agents: (1) dithioesters,where Z is aryl or alkyl, (2) trithiocarbonates where Z is a substitutedsulphur, (3) dithiocarbonates (xanthates), where Z is substitutedoxygen, and (4) dithiocarbamates, where Z is substituted nitrogen.

In one embodiment, the functionalized amide-containing initiator is anNHS-functionalized amide containing ATRP initiator, and morespecifically refers to N-2-bromo-2-methylpropanoyl-β-alanineN′-oxysuccinimide ester (II).

In another embodiment, the functionalized amide-containing initiator isan NHS-functionalized amide-containing ATRP initiator, specificallyreferring to N-2-chloro-propanoyl-β-alanine N′-oxysuccinimde ester(III), which has Cl as the halogen and one less methyl group next to thehalogen than NHS initiator (II).

The active esters R₂ in structure (I) may be selected from thefollowing:

N-oxysuccinimide ester

nitrophenyl ester

pentahalophenyl ester, wherein X is F or Cl;

1-oxybenzotriazole ester; and,

2-oxy-4,6-dimethoxy-1,3,5-triazine ester.Proteins

Proteins are comprised of fewer than one hundred to thousands of aminoacid residues linked by peptide bonds linearly and/or branched, andfolded over in three-dimensional configurations. The configuration ofthe protein determines function. Enzymes, for example, function asbiological catalysts that may increase the rate of a biological reactionby 10⁶ to 10¹⁴ fold. Most enzymes are reactive under mild physiologicalconditions. The configuration of an enzyme, and therefore, the positionof available binding sites contribute to the specificity and selectivityof enzymes. Enzymes have an active binding site to receive and bind witha substrate, such as another molecule, to form enzyme-substratecomplexes. Upon binding, the enzyme catalyzes the relevant reaction toproduce the end product of the catalyzed reaction. Enzymes interact withtheir substrates and targets by removing them from a solvent, binding,reacting and then returning products to solution. In nature, there arecomplex protein-protein interactions to achieve modification of thepolypeptide to form the final protein structure with its specificfunction.

Modification of proteins may be achieved biologically by randommutation, or by cloning/expression, expression systems, biodiversitymining, site directed mutagenesis, or directed evolution. These methods,however, often yield only incremental improvements, are difficult andexpensive to scale, require long development times, and often onlyprovide situational solutions. Modification of proteins may be achievedchemically by activity in organic solvents, immobilization,cross-linking, conjugation to antibodies, peptide or small moleculeconjugation, encapsulation, or phase separation using micelles. Thesetechniques are unsatisfactory because they often result in loweractivity, often only provide situational solutions, and often havecompatibility issues in vivo.

The invention described herein offers an alternative route to proteinmodification using polymer based protein engineering. Polymers arerationally designed to specifically alter a protein function. In oneembodiment of the method of the invention, an enzyme, such aschymotrypsin, lysozyme, β-Galactosidase, carbonic anhydrase, glucoseoxidase, laccase, or acetylcholinesterase, or any protein having surfacereactive amino acid side chains that can be covalently coupled to theactive ester functionalized amide-containing initiator, is reacted withthe functionalized amide-containing initiator to form an initiatormodified protein, or initiator modified enzyme, referred to herein as amacroinitiator. Exemplary initiators include, for example, anN-functionalized propanoyl-β-alanine N′-oxysuccinimide ester. Thefunctional group for binding with the surface reactive amino acid on theprotein is the active ester. The functional group that reacts with themonomers in the polymerization may be a halogen, such as Br, F, or Cl,or a chain transfer agent. The active ester functionalizedamide-containing initiator is immobilized to the amines on the surfaceof the protein in aqueous solution. Using the “grafting from” approach,the macroinitiator is reacted with monomers of choice in a controlledradical polymerization reaction, such as atom transfer radicalpolymerization (ATRP) or reversible-addition fragmentation chaintransfer (RAFT), to grow the polymer chains on each active site wherethe amide-containing initiator was immobilized. The result is aprotein-polymer conjugate that provides a bioactive molecule havingdesired properties. The function of the originating protein will bemodified by the properties of the conjugated polymers.

In various embodiments, the functionalized amide-containing initiator isformed by the introduction of amide group(s) in the initiator reagent toincrease hydrophilicity. One embodiment of the method proceeds, ingeneral, as follows:

1. immobilization of active ester functionalized amide-containinginitiator on a protein surface, for example, by mixing the protein ofchoice and the active ester functionalized amide-containing initiator ina buffer (e.g., 100 mM Na phosphate, pH 8-9) and stirring at, forexample, 4° C. for about 3 hours, or a sufficient time to allow theimmobilization to occur.

2. isolating the protein-initiator conjugate, for example, by removingany unreacted or unattached small compounds, such as any free initiator,by for example, using a dialysis membrane. The step may also includelyophilizing, and characterizing the protein-initiator conjugate.

3. growing polymer from the protein by “grafting from” using acontrolled radical polymerization procedure, such as ATRP or RAFT, orany of the other well documented controlled radical polymerizationprocedures. This step may include mixing the protein-initiator conjugateand monomer(s) in buffer, charging (bubble) Ar or N₂ to remove oxygen;adding a ligand in Ar or N₂ charged water, then adding a catalyst (forexample, in an ATRP procedure, a copper catalyst, such as Cu(I)) with Aror N₂ bubbling for oxygen removal. The copper-ligand catalyst solutionis transferred to the protein/monomer solution using, for example, an Aror N₂ charged syringe. The combined solutions are stirred at about 4-25°C. for a sufficient time to allow polymerization to proceed, typicallyabout 4 hours or overnight.

4. isolating the protein-polymer conjugate, by for example, using adialysis membrane and optionally refrigerating overnight to removecatalyst, such as any copper-ligand catalyst, and any unreactedmonomer(s). The method may also include lyophilizing, and characterizingthe protein-initiator conjugate.

Chymotrypsin (CT) is a serine protease that acts in the small intestine,and was selected for use as the exemplary protein in the series ofexperiments described herein due to the large amount of informationavailable on enzyme activity and stability at a wide range of pH andtemperature. (Hedstrom, L. Chem. Rev. 2002, 102, 4501; Kumar, A.;Venkatesu, P. Chem. Rev. 2012, 112, 4283). Chymotrypsin contains 14surface lysine's, as well as the N-terminus, which allows for highdensity attachment of polymer around the protein when using the ATRPinitiator described herein that reacts preferentially with primaryamines.

In addition, chymotrypsin based protein-polymer conjugates could be usedto treat exocrine pancreatic insufficiency (Larger, E.; Philippe, M. F.;Barbot-Trystram, L.; Radu, A.; Rotariu, M.; Nobécourt, E.; Boitard, C.Diabetic Medicine 2012, 29, 1047), but the enzyme would have to firstsurvive passage through the stomach and into the small intestine.Previously, β-galactosidase (see Turner, K. M.; Pasut, G.; Veronese, F.M.; Boyce, A.; Walsh, G. Biotechnol. Lett. 2011, 33, 617) (for lactoseintolerance) and proline specific endopeptidases (see Fuhrmann, G.;Grotzky, A.; Lukic, R.; Matoori, S.; Luciani, P.; Yu, H.; Zhang, B.;Walde, P.; Schluter, A. D.; Gauthier, M. A.; Leroux, J. C. Nat. Chem.2013, 5, 582) (for coeliac disease) have been modified with polymers tostabilize proteins with varying success.

Other proteins may be used for the macroinitiator. For example,Lysozyme, β-Galactosidase, carbonic anhydrase, glucose oxidase, laccase,and acetylcholinesterase have each been successfully modified byattachhment to one of the NHS-functionalized initiators described hereinto form various embodiments of the protein initiator conjugate of theinvention. Most have additionally been modifed with polymer attachmentusing the growing from method described herein to form variousembodiments of the protein-polymer conjugates. In further examples, auricase based protein-polymer conjugate may be synthesized and could beused for treatment of chronic gout. A PEGylated uricase drug(PEGloticase) currently approved by FDA for treatment of chronic goutmust be injected intravenously. It lowers uric acid concentration inblood stream.

Polymers

Polymer choice is crucial in predicting efficacy of polymer conjugationfor the desired purpose. The choice of polymer to bind to the protein ofchoice using an embodiment of the methods described herein will bedictated by the activity sought to be manipulated. Protein function canbe altered by polymer based protein engineering using synthetic orbiologically inspired monomers. For example, choosing one or morepolymers for stabilization in the GI tract, will allow ingestion of thebioconjugate for in vivo delivery to a desired site where the proteinmay have a role. The polymer-based protein engineering approach withresponsive polymers as described herein will be an improvement toconventional treatments because, in one embodiment, the protein-polymerconjugate produced by the methods described allows conjugation withpolymers that will modify the enzyme function to increase stability andpermit transport across the intestinal wall into bloodstream. Thepolymer additions protect the enzyme through low pH stomach conditions,as demonstrated in vitro for CT-pSBAm-block-pNIPAm and CT-pQA conjugatesdescribed in more detail in the Experimental series herein. Also,poly(acrylic acid) which collapses at low pH may give increasedstability to the enzyme in the stomach. Piperazine may be used as thepolymer to increase permeation across the intestinal membrane.

The results seen in the series of experiments described herein may beapplied not only to chymotrypsin, but to other proteins, particularlyenzymes, having accessible surface reactive amino acid side chains thatcan covalently couple to the active ester functionalizedamide-containing initiator molecule. Two polymers, pSBAm and pNIPAm,were chosen to study the effect of phase transitions at both high andlow temperature on CT bioactivity. The LCST temperature of pNIPAm isbetween room temperature and body temperature. Thus, it is a goodcandidate to incorporate into materials that might need to besynthesized in aqueous solution at room temperature, but then changebehavior once in the body, such as an enzyme targeted to fat tissuewhere it would likely need to be hydrophobic. Attaching a UCST polymerto an enzyme can potentially increase stability at low temperature, andincrease long term storage time before use. A protein-polymer conjugateincorporating both of these polymers, which is described herein, couldserve both purposes. In addition, the unique geometry of both UCST andLCST containing polymers in the same chain allowed for the examinationof the interaction between each polymer block and CT at different phasetransition temperatures. Stimuli responsive protein-polymer conjugatesthat respond to one stimulus often show slightly different behavior thanfree polymer because of interactions with or shielding by the protein.It was hypothesized that temperature responsive properties could bealtered by the enzyme as well as another polymer block that doesn'trespond to stimuli (similar to pNIPAAm-b-PAA, see Kulkarni, S.; Schilli,C.; Grin, B.; Muller, A. H. E.; Hoffman, A. S.; Stayton, P. S.Biomacromolecules 2006, 7, 2736) or that responds to a differentstimulus. Thus, a chymotrypsin protein-polymer conjugate was designed toeasily examine this hypothesis as well as the effect of polymerconjugation of enzyme bioactivity at multiple stimuli (high and lowtemperature).

EXPERIMENTAL SECTION Materials and Methods

Materials

α-Chymotrypsin (CT) from bovine pancreas (type II),2-Bromo-2-methylpropionyl bromide, pepsin from porcine stomach mucosa,Aprotinin (Bovine, recombinant, expressed in Nicotiana (tobacco)),Bowman-Birk chymotrypsin inhibitor from glycine max (soybean), p-toluenesulfonic acid, β-alanine, N-hydroxysuccinimide,N,N′-diisopropylcarbodiimide, copper (I) bromide, copper (I) chloride,1,1,4,7,10,10-Hexamethyltriethylenetetramine (HMTETA),2-(dimethylamino)ethyl methacrylate (DMAEMA, passed over a column ofbasic alumina prior to use),N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitoroanilide (Suc-AAPF-pNA),bicinchoninic acid (BCA) solution, copper (II) sulfate solution,dichloromethane, ethyl acetate, 2-propanol, diethyl ether, n-hexane and[2-(Methacryloylamino)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide)(sulfobetaine methacrylamide),(12-Meth-acryloyloxy)ethyl[dimethyl-(3-sulfopropyl ammonium hydroxide)(DMAPS) were purchased from Sigma Aldrich (St Louis, Mo.) and usedwithout further purification. N-isopropylacrylamide was purchased fromSigma Aldrich (St. Louis, Mo.) and purified by recrystallization usinghexane. Tris[2-(dimethylamino)ethyl]amine (Me6TREN) was synthesized asdescribed by Ciampolini, M.; Nardi, N. Inorg. Chem. 1966, 5, 41.Quaternary ammonium (QA) monomer (2-(dimethylethylammonium)ethylmethacrylate) was synthesized according to modified procedure reportedby Tsarevsky and co-workers (Tsarevsky, N. V.; Pintauer, T.;Matyjaszewski, K. Macromolecules 2004, 37, 9768.) (see details inExperimental Series III below). Dialysis tubes (molecular weight cutoff, 25-, 15- and 1-kDa (Spectra/Pore, Spectrum Laboratories Inc., CA))were purchased from Fisher Scientific (Pittsburgh, Pa.).

Measurements

¹H-NMR spectra were recorded on a spectrometer (300 MHz, Bruker Avance)in the NMR facility located in Center for Molecular Analysis, CarnegieMellon University, Pittsburgh, USA with Deuterium oxide (D₂O), DMSO-d₆,and CDCl₃. Routine FT-IR spectra were obtained with a Nicolet Avatar 360FT-IR spectrometer (Thermo). UV-VIS spectra were obtained and used forenzyme activity determination using a UV-VIS spectrometer (Lambda 2,PerkinElmer) with a temperature-controlled cell holder. Number andweight average molecular weights (M_(n) and M_(w)) and thepolydispersity index (M_(w)/M_(n)) were estimated by gel permeationchromatography (GPC) on a Water 2695 Series with a data processor,equipped with three columns (Waters ultrahydrogel Linear, 500 and 250),using 100 mM sodium phosphate buffer with 0.2 vol % trifluoroacetic acid(pH 2.5) as an eluent at a flow rate 1.0 mL/min and and[2-(Methacryloylamino)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide)(sulfobetaine methacrylamide, with detection by a refractive index (RI)detector. Polyethylene glycol standards were used for calibration.Melting points (mp) were measured with a Laboratory Devices Mel-Temp.

Experimental Series I Polymer Based Protein Engineering to RationallyTune Enzyme Activity, pH Dependence and Stability

Procedure for Synthesis of NHS-Functionalized ATRP Initiator.

N-2-bromo-2-methylpropanoyl-β-alanine N′-oxysuccinimide ester, theNHS-functionalized amide containing ATRP initiator described herein, wassynthesized as follows. A mixture of 2-bromo-2-methylpropionyl bromide(12.4 mL, 100 mmol) and dichloromethane (50 ml) was slowly added intothe solution of β-alanine (8.9 g, 100 mmol) and sodium hydrogencarbonate (21 g, 250 mmol) in de-ionized water (200 mL) at 0° C. thenthe mixture was stirred at room temperature for 2 h. The water phase waswashed with dichloromethane (100 mL×3) and adjusted to pH 2 with 1.0 NHCl aq. at 0° C. The product was extracted with ethyl acetate (150mL×6). The organic phase was dried with MgSO₄ and evaporated to removesolvent. N-2-bromo-methylpropionyl-β-alanine was isolated byrecrystallization from mixture of diethyl ether and n-hexane (1/9 volumeratio). N,N′-diisopropylcarbodiimide (2.8 g, 22 mmol) was slowly addedto the solution of N-2-bromo-2-methylpropionyl-β-alanine (4.8 g, 20mmol), and N-hydroxysuccinimide (2.5 g, 22 mmol) in dichloromethane (200mL) at 0° C. The mixture was stirred at room temperature for 4 h. Afterfiltering out the precipitated urea, the solution was evaporated toremove solvents. N-2-bromo-2-methylpropionyl-β-alanine N′-oxysuccinimideester (1) was purified by recrystallization from 2-propanol with a yieldof 5.7 g (85%) mp 120-124° C. The chemical structure was confirmed by¹H-NMR and IR.

Preparation of the CT-Initiator Conjugate.

CT (1.0 g, 0.56 mmol of amine groups contained) was dissolved in 100 mMsodium phosphate buffer (pH 8.0) at 0° C. After adding theNHS-functionalized ATRP initiator (619 mg, 1.85 mmol), the mixture wasstirred in a refrigerator for 3 h and the CT-initiator conjugate wasisolated by dialysis using a 15-kDa molecular weight cut-off dialysistube in de-ionized water in a refrigerator for 24 h and thenlyophilized.

Preparation of CT-PDMAEMA Conjugates.

A solution of DMAEMA (169 μL, 1.0 mmol for sample conjugate 1 (C1); 337μL, 2.0 mmol for C2; 675 μL, 4.0 mmol for C3; 1.35 mL, 8.0 mmol for C4)and CT-initiator conjugate (100 mg, 0.046 mmol of initiator groups) inde-ionized water (30 mL) was sealed and bubbled with Argon in an icebath for 50 min. Deoxygenated catalyst solutions of HMTETA (55 μL, 0.2mmol) and Cu(I)Br (29 mg, 0.2 mmol) in de-ionized water (10 mL) was thenadded to the conjugation reactor under Argon bubbling. The mixture wassealed and stirred for 18 h at 4° C. to avoid self-polymerization of theDMAEMA. CT-PDMAEMA conjugates were isolated by dialysis with a 25-kDamolecular weight cut-off dialysis tube in de-ionized water in arefrigerator for 24 h, and then lyophilized.

Cleavage of the Grafted PDMAEMA from the Conjugate.

CT-PDMAEMA conjugate (10 to 20 mg) and 6 N HCl aq. (2 to 3 mL) wereplaced in a hydrolysis tube. After three freeze-pump-thaw cycles thehydrolysis was performed at 110° C. for 24 h in vacuum. The cleavedpolymer was isolated by dialysis using a 1-kDa molecular weight cut offdialysis tube in de-ionized water and was then lyophilized. Themolecular weight of the cleaved polymer was measured by GPC.

Determination of Molecular Weight of the Prepared Conjugates.

Molecular weights of the prepared CT-PDMAEMA conjugates were calculatedfrom estimated molecular weight of cleaved PDMEAME from the conjugate.BCA and absorption assays were also carried out to determine molecularweight of the conjugates. Detailed procedures are provided in theSupplementary Materials section.

Matrix-Assisted Laser Desorption Ionization Time-of-Flight Spectrometry(MALDI-TOF MS).

The molecular weight of the CT-ATRP initiator conjugate was estimatedwith a Perseptive Biosystems Voyager Elite MALDI-TOF spectrometer inCenter for Molecular Analysis, Carnegie Mellon University, Pittsburgh,Pa. The operation of MALDI-TOF spectrum was performed as describedpreviously. (Lele, B. S.; Murata, H.; Matyjaszewski, K.; Russell, A. J.Biomacromolecules 2005, 6, 3380-3387).

Dynamic Light Scattering (DLS).

The DLS data were collected on a Malvern Zetasizer nano-ZS, which waslocated in the Department of Chemistry, Carnegie Mellon University,Pittsburgh, USA. The concentration of the sample solution was kept at1.0 mg/mL. The hydrodynamic diameter of the native CT and the conjugatewas measured three times (12 run to each measurement) at various pH's.

Low Critical Solution Temperature (LCST) Point Measurements.

The CT-PDMAEMA conjugate (1.0 mg/mL in 100 mM sodium phosphate buffer pH8.0) was placed in a glass cuvette, in a UV-VIS spectrometer and theincrease in absorption at 490 nm was monitored as function oftemperature (0.5-1.0° C./min). The LCST was determined as the initialonset of a sharp increase in absorbance at 490 nm (FIG. 9).

Enzyme Kinetic Assay.

N-Succinyl-Ala-Ala-Pro-Phe p-nitroanilide (0 to 50 μL of 9.60 mM inDMSO) was added to sodium phosphate buffer (990 to 940 μL of 100 mM, pH5-10). Native CT or conjugates solution (10 μL of 4.0 μM) was added tothe substrate solution. The initial rate of hydrolysis of the peptidesubstrate was monitored by recording the increase in absorption at 412nm using a UV-VIS spectrometer. The Michaelis-Menten parameters(k_(cat), K_(m) and k_(cat)/K_(m)) were determined by non-linear curvefitting (equation for Michaelis-Menten parameters) of plots of initialrate versus substrate concentration using the Enzfitter software.

Stability Study.

Native CT and the prepared conjugates (1.5 to 2.0 mL of 40.0 μM) wereincubated in 100 mM sodium phosphate buffer at different pH's at 40° C.Aliquots (20 μL) were removed and added into sodium phosphate buffer(180 μL, 100 mM, at 0° C.). The residual activity was calculated as aratio of initial rates of hydrolysis reaction at given incubation timeover the initial activity, which monitored spectrophotometrically asdescribed above. Half-lives for enzyme longevity were estimated bynon-linear curve fitting (equation for single exponential decay) usingEnzfitter.

Results and Discussion

Synthesis and Attachment of a Water Soluble ATRP Initiator

A clear gap in current protein engineering methods is the availabilityof a water-soluble protein-reactive ATRP initiator that could be used toavoid the exposure of proteins to mixtures of water and organic solventsduring PBPE. A novel NHS-functionalized amide-containing ATRP initiatorwas designed and an example is illustrated in the schematic of FIG. 1.Results from ¹H-NMR and MALDI-TOF MS demonstrated that that 85% of theCT surface lysines reacted with the initiator (12 of 14 amine groups),providing one initiating point per 5 nm² of enzyme surface and isillustrated in FIG. 2. Since the initiator density was less than highestpractical grafting-from density of PDMAEMA on surfaces (0.6 to 0.99nm⁻²), it was expected every initiator point to be fully active.Further, this chain density should not lead to early termination and ishigher than any other reported approach to protein-initiated ATRP by anorder of magnitude. This implies that by simply varying thestoichiometry of the enzyme and initiator the number of sites ofattachment can be fine-tuned to exhibit desired properties.

In one embodiment, polymer-based protein engineering (PBPE) wasperformed with DMAEMA and CT in the presence of copper (I) bromide andHMTETA in buffer (FIG. 1). The chain length of the extending polymericchains was dependent on the initial molar concentration of DMAEMA andthe results are illustrated in Table 1.

TABLE 1 Impact of polymerization conditions on the size of CT-PDMAEMAconjugates. Polymerization Cleaved Mw of Conjugate (kDa) Concentrations¹Yield² Polymer³ R_(g) ⁴ R_(g)/ Cleaved D_(h) ⁸ LCST⁹ Sample [I]₀:[M]₀(%) M_(n) (M_(w)/M_(n)) (nm) D⁵ Polymer⁶ BCA⁷ Absorption⁷ (nm) (° C.) C11:21 63  4,800 (1.51) 2.8 1.2 85.7 53.8 66.7 13.0± 51 2.3 C2 1:42 65 9,300 (1.55) 3.8 1.7 139.7 131.4 120.0 18.6 ± 49 3.3 C3 1:83 82 14,700(1.52) 4.8 2.2 204.5 196.2 202.7 25.2 ± 48 3.8 C4  1:163 95 23,100(1.57) 6.1 2.7 305.3 350.3 354.2 34.3 ± 47 3.3 ¹The enzyme was modifiedwith 12 initiator units per molecule and the initial initiatorconcentration ([I]₀) was used to calculate the initial concentrations ofcopper bromide and HMTETA so as to achieve a starting ratio of 1:5:5([I]₀:[Cu(I)Br]₀:[HMTETA]₀) in deionized water at 4° C. ²The yield wasdefined as the percentage of the total weight of lyophilized CT-PDMAEMAconjugate/total weight of initiator-modified chymotrypsin. ³GraftedPDMAEMA was cleaved by vacuum hydrolysis method using 6N HCl at 110° C.for 24 h. Cleaved polymer was isolated by dialysis and the molecularweight of cleaved polymer was estimated by GPC. ⁴The radius of gyrationof the polymer in the solution (R_(g)) was assumed to be 0.5N^(0.5).⁵The average distances between polymer chains (D) was assumed to be =2.2nm (1/σ⁻²). ⁶The molecular weight of conjugates was calculated from themeasured molecular weight (M_(n)) of the cleaved polymer (assuming 12chains of equal length) and the initial molecular weight of theinitiator-modified enzyme. ⁷The details of the BCA and absorptionmethods for molecular weight determination are described in theSupplementary Materials. ⁸The hydrodynamic diameter (D_(h)) of theCT-PDMAEMA conjugate was determined by dynamic light scattering at 1.0mg/mL in 100 mM sodium phosphate (pH 7.0) at 25° C. The D_(h) of nativeCT was 4.4 ± 1.3 nm. ⁹The LSCT was measured with a UV-VISspectrophotometer at 1.0 mg/mL in 100 mM sodium phosphate buffer (pH8.0).

A particular challenge in PBPE is to determine the actual chain lengthof the polymers. Polymer chains were cleaved from the conjugate byvacuum hydrolysis in 6 N HCl aq at 110° C., followed by isolation withdialysis, and then determined chain length by GPC. The data isillustrated in FIG. 3 and Table 2, and demonstrates rational control ofchain length and that all chains grow at the same rate resulting in arelatively low PDI.

TABLE 2 Properties of cleaved PDMAEMA from the conjugates Cleavedpolymer¹ Degree of Sample M_(n) (M_(w)/M_(n)) polymerization² R_(g) ³CP1  4,800 (1.51) 30.6 2.8 CP2  9,300 (1.55) 59.2 3.8 CP3 14,700 (1.52)93.6 4.8 CP4 23,100 (1.57) 147.1 6.1 ¹Molecular weight and the polymerdispersity index of cleaved pDMAEMA from the conjugate was determined byGPC. ²Degree of polymerization = M_(n) of cleaved pDMAEMA/M_(w) ofDMAEMA unit (157 g/mol). ³Radius of gyration of cleaved pDMAEMA R_(g) =0.5N^(0.5), N is degree of polymerization ref).

In another example, the conformation of the grafted polymer chains andits impact on CT activity and stability was explored. Polymers onsurfaces switch between “mushroom” and “brush” regime as a function ofsurface density and chain length. (Brittain, W. J.; Minko, S. J. Polym.Sci.: Part A: Polym. Chem. 2007, 45, 3505-3512). For conjugate C1 and C2the calculated R_(g)/D values imply that the polymer had a “mushroom”structure whereas in C3 and C4 the surface density and length wouldyield a “brush” structure. (Dong, Z.; Wei, H.; Mao, J.; Wang, D.; Yang,M.; Bo, S.; Ji, X. Polymer 2012, 53, 2074-2084; Uchida, E.; Ikada, Y.Macromolecules 1997, 30, 5464-5469). The hydrodynamic diameters (D_(h))of the conjugates were determined using dynamic light scattering (DLS).As expected, D_(h) increased with the length of the grafted PDMAEMA.This result was consistent with theoretical studies relating layerthickness to polymer length on spherical surfaces. (Wijmans, C. M.;Zhulina, E. B. Macromolecules 1993, 26, 7214-7224). Thus, PBPE usingATRP provided us with rational control of enzyme-polymer conjugate size.

Tailoring pH-Dependence of Enzyme Activity using PBPE

At pH's below the pK_(a), PDMAEMA chains stretch and swell as result ofelectrostatic repulsion between the protonated tertiary amine groups. AtpH's above the pK_(a), the tertiary amine groups of the PDMAEMA aredeprotonated and hydrophobic, resulting in collapsed chains. Theinfluence of pH on the size and catalytic activity of the CT-PDMAEMAbioconjugates was investigated in another example. Conjugate diameterincreased at low pH (pH 5 to 7), indicating that the grafted PDMAEMAbehaved as the polymer did in solution, with the same pK_(a) (FIG. 4c ).(van de Wetering, P.; Zuidam, N. J.; van Steenbergen, M. J.; van derHouwen, O. A. G. J.; Underberg, W. J. M.; Hennink, W. E. Macromolecules1998, 31, 8063-8068). The largest decrease of the particle size as afunction of increased pH (30% size reduction) was observed on theconjugate with the longest chains (FIG. 4b ).

PBPE would be most useful if one could rationally tailor enzymaticactivity by designing the properties and length of the polymer chains.The activity of chymotrypsin is dependent on a catalytic triad that isstabilized by a charge relay network, and therefore charged polymerconjugates may influence activity. Additionally, the conformation of thepolymer chains may influence the ability of substrate to bind to theenzyme. The complete kinetic behavior of the CT-catalyzed hydrolysis ofthe model substrate succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanylp-nitroanilide (Suc-AAPF-pNA) was measured as a function of pH (Table3).

TABLE 3 Polymerization conditions and property of free PDMAEMAPolymerization Degree of Condition¹ Yield² Poly- sample [M]₀/[I]₀ (%)M_(n) (M_(w)/M_(n))³ merization⁴ R_(g) ⁵ F1  25/1 70  4,600 (1.71) 29.32.7 F2  50/1 73  9,600 (1.53) 61.1 3.9 F3 100/1 83 13,500 (1.56) 86.04.6 F4 200/1 88 23,000 (1.73) 142.0 6.0 ¹[I]₀:[Cu(I)Br]₀:[HMTETA]₀1:1.5:1.5, DI water, 18 h. ²Yield (%) = total weight of lyophilized freePDMAEMA/total weight of loaded initiator and DMAEMA × 100. ³Molecularweight of free PDMAEMA was estimated by GPC. ⁴Degree of polymerization =M_(n) of cleaved pDMAEMA/M_(w) of DMAEMA unit (157 g/mol). ⁵R_(g)(radius of gyration of the polymer in the solution) = 0.5N^(0.5).

The data revealed that above pH 6 the turnover number, k_(cat), wasinfluenced only when the conjugate moved from the “mushroom” (C1 and C2)to the “brush” conformation (C3 and C4). For PEGylated CT, k_(cat) was afunction of chain density but not chain length. (Rodriguez-Martinez, J.A.; Rivera-Rivera, I.; Solá, R. J.; Griebenow, K. Biotechnol. Lett.2009, 31, 883-887) It was observed that above pH 9 the k_(cat)'s for allthe conjugates were similar; above pH 9 the grafted PDMAEMA on all ofconjugates would be deprotonated. Below pH 6, a significant increase ink_(cat) for the conjugates was observed relative to native enzyme. Thisresult is interpreted as the protonated PDMAEMA stabilizing thenegatively charged carboxyl group of Aspartate 102 in the catalytictriad of the CT, thereby shifting the pH-dependence of the enzyme so asto increase activity at low pH. Similarly, site-directed mutagenesis onserine proteases has been shown to tailor rationally pH-dependence byaltering enzyme surface charge. (Russell, A. J.; Thomas, P. G.; Fersht,A. R. J. Mol. Biol. 1987, 193, 803-813; 21. Russell, A. J.; Fersht, A.R. Nature 1987, 328, 496-500).

Once a substrate has bound to CT, the charge relay system governs theactivity of the enzyme. PBPE's impact on the binding affinity between CTand its model substrate was examined in another example. The Michaelisconstant, K_(M), for all the conjugates at a range of pH's wasdetermined. Below pH 8 substrate binding was improved by PBPE, resultingin a lowered K_(M) (FIG. 5b ). Within this pH range, the PDMAEMAconjugates were, of course, charged, with long-separated chains.PEGylation of CT is known to increase K_(M) but the structure of PEG ona surface cannot be tuned by changing pH. The results described hereinsuggest that the well separated ATRP-generated chains did not interferewith the natural state of the active site and at low pH the enzymeretained its native conformation more effectively as a result ofincreased surface charge. Above the pK_(a) of PDMAEMA it was observedthat the K_(M)s of the conjugates relative to native CT weresignificantly increased. Without wishing to be bound by theory, it ishypothesized that this reduction of affinity of the substrate at higherpH was because the substrate binding to the active site was inhibited byhydrophobic-hydrophobic interaction between the substrate anddeprotonated grafted PDMAEMA chains. Also, collapsed PDMAEMA chainswould be expected to sterically hinder the substrate binding pocket ofthe conjugate. A significant increase in K_(M) for conjugates C3 and C4was observed, which have longer grafted PDMAEMA chains at pH 10 (FIG. 5b). Thus, the impact of PBPE methods of the present invention onCT-substrate binding was tunable and permitted reliable prediction ofprotein-polymer conjugate function.

Combining the data and understanding for the impact of PBPE on k_(cat)and K_(M) separately, it was observed higher catalytic efficiency(k_(cat)/K_(M)) at lower pHs (FIG. 5c ). PBPE led to an almost ten-foldincrease in catalytic efficiency of the conjugates at pH 5. Forconjugates C1 and C2, which had shorter PDMAEMA chains, the catalyticefficiency was not significantly changed above pH 8.0. Conjugates C3 andC4, however, did exhibit an approximately 50% reduction in catalyticefficiency above pH 8.0. This pH-dependence of enzyme activity for longchain modified enzyme was undoubtedly driven by the K_(M) effectsdescribed above (FIG. 5b ).

Impact of Polymer Conformation Temperature Dependence on PBPE-CTActivity

PDMAEMA is also a thermo-responsive polymer with an LCST that isdependent on pH, ionic strength, and molecular weight. Linear andstar-shaped PDMAEMAs have LCST's of approximately 50° C. at pH 8. TheLCST drops as chain length increases. Below the LCST, PDMAEMA chains ofsufficient lengths are hydrated and expanded. (Dong, Z.; Wei, H.; Mao,J.; Wang, D.; Yang, M.; Bo, S.; Ji, X. Polymer 2012, 53, 2074-2084) Itwas determined that the bioconjugates would also change in conformationand relative activity with changes in temperature. The PBPE conjugatesshowed cloud points between 47-51° C. in the buffer at pH 8.0,exhibiting a drop in LCST with increasing chain length (Table 1 and FIG.6). Repeated cloud point transitions did not alter enzyme activity insolution.

In one embodiment, the effect of PBPE on the temperature dependence ofactivity was determined by measuring the complete kinetic profiles ofthe native and modified enzymes with respect to the hydrolysis of theSuc-AAPF-pNA in 100 mM sodium phosphate buffer (pH 8.0) between 25 and53° C. (FIG. 7 and Table 4).

TABLE 4 Michaelis-Menten parameters of hydrolysis of N-Suc-AlaAlaProPhe-pNA under different pH buffer solution. sample pH 5.0 pH6.0 pH 7.0 pH 8.0 pH 9.0 pH 10.0 native k_(cat) 2.28 ± 0.36 12.13 ±21.54 ± 26.67 ± 29.49 ± 27.69 ± 0.57 0.63 1.47 2.00 1.11 K_(m) 212.2 ±188.9 ±  84.1 ±  55.2 ± 103.6 ± 257.7 ± 67.2 18.3 7.5 9.7 17.6 19.2k_(cat)/ 0.011 ± 0.064 ± 0.256 ± 0.483 ± 0.284 ± 0.107 ± K_(m) 0.0040.007 0.024 0.089 0.052 0.009 C1 k_(cat) 5.90 ± 0.30 14.90 ± 22.69 ±32.82 ± 32.85 ± 29.49 ± 0.63 0.74 0.75 1.54 2.64 K_(m) 62.5 ± 9.6  65.7± 8.2  52.2 ±  39.8 ± 105.1 ± 318.7 ± 6.1 3.3 12.4 49.4 k_(cat)/ 0.094 ±0.227 ± 0.435 ± 0.825 ± 0.313 ± 0.092 ± K_(m) 0.015 0.030 0.053 0.0700.040 0.017 C2 k_(cat) 5.44 ± 0.40 14.51 ± 21.08 ± 29.49 ± 33.85 ± 27.18± 0.60 0.34 0.70 0.75 1.84 K_(m) 57.9 ± 13.2  57.2 ± 42.5 ± 2.6  36.8 ±107.6 ± 283.1 ± 7.4 3.2 6.0 34.7 k_(cat)/ 0.094 ± 0.254 ± 0.496 ± 0.801± 0.313 ± 0.096 ± K_(m) 0.023 0.035 0.032 0.073 0.019 0.013 C3 k_(cat)4.97 ± 0.38 10.85 ± 15.96 ± 20.82 ± 24.03 ± 21.31 ± 0.20 0.33 0.42 0.752.63 K_(m) 88.2 ± 17.8 51.9 ± 3.1  34.3 ±  34.7 ± 108.3 ± 408.6 ± 2.92.3 8.4 81.2 k_(cat)/ 0.056 ±  0.209 ± 0.013 0.466 ± 0.600 ± 0.222 ±0.052 ± K_(m) 0.012 0.041 0.041 0.019 0.012 C4 k_(cat) 3.36 ± 0.16  8.38± 11.96 ± 17.30 ± 19.21 ± 15.46 ± 0.31 0.26 0.80 0.80 1.29 K_(m) 71.4 ±9.6   39.4 ±  27.1 ±  38.0 ± 112.2 ± 368.2 ± 5.3 2.8 6.5 11.5 50.8k_(cat)/ 0.047 ± 0.213 ± 0.442 ± 0.455 ± 0.171 ± 0.042 ± K_(m) 0.0070.030 0.046 0.081 0.019 0.007 Units are as follows: k_(cat), sec⁻¹;K_(m), μM; k_(cat)/K_(m), sec⁻¹ · μM⁻¹.

As was the case for pH-induced changes in bioconjugate conformation, thek_(cat)'s of the bioconjugates did not exhibit unusual changes at theLCST (FIG. 7a ). Some indication was observed that as polymer chainlength increased, the degree of temperature dependence of k_(cat)decreased, but this difference was not statistically significant. Astemperature increased and the polymer became more “mushroom-like”, beingtightly woven around the protein surface, a significant increase inK_(M) relative to that of the native enzyme (FIG. 7b ) was observed.Although only three temperatures were probed, the response of K_(M) totemperatures above the LCST resembled the impact of pH>8.0 on bindingaffinity. Once again, the hydrophobic characteristics of the peptidesubstrate were likely interacting with the now hydrophobic PDMAEMA chainthat was also sterically hindering access to the active site of the CT(FIG. 7b ). Consequently, catalytic efficiency (k_(cat)/K_(M)) of thePBPE-synthesized conjugate was significantly decreased at temperaturesabove the LCST of the grafted PDMAEMA (FIG. 7c ).

Functional Stability of CT-PDMAEMA Conjugates

CT inactivation can occur through unfolding and/or autolysis. Thepolymer component of the PBPE conjugates may stabilize the enzyme byincreasing the energy needed to unfold the protein and also byinhibiting autolysis directly. Since the autolysis reaction of proteasesis favored when the enzyme concentration is high, (Yang, Z.; Domach, M.;Auger, R.; Yang, F. X.; Russell, A. J. Enzyme Microb. Technol. 1996, 18,82-89) stability at relatively high enzyme concentration (40 μM) wasinvestigated. At 40° C. the residual activity of the enzyme was measuredas a function of pH and shown in FIG. 8, and the first order rateconstant for deactivation and half-life were calculated (Table 5).

TABLE 5 pH-Dependence of irreversible enzyme inactivation for native CTand PBPE conjugates. pH Native CT C1 C2 C3 C4 7.0 k_(d) (1/s) 2.69 ×10⁻⁵ 9.91 × 10⁻⁷ 8.13 × 10⁻⁷ 9.30 × 10⁻⁷ 1.06 × 10⁻⁶ t_(1/2) (h) 7.15 ±0.91 194 ± 41 237 ± 58 207 ± 50 183 ± 14  8.0 k_(d) (1/s) 9.88 × 10⁻⁵5.22 × 10⁻⁶ 6.38 × 10⁻⁶ 9.91 × 10⁻⁶ 2.78 × 10⁻⁵ t_(1/2) (h) 1.95 ± 0.2536.87 ± 30.19 ± 19.43 ± 6.92 ± 1.73 6.46 6.83 4.92 9.0 k_(d) (1/s) 1.26× 10⁻⁴ 7.14 × 10⁻⁵ 6.82 × 10⁻⁵ −7.24 × 8.99 × 10⁻⁵ 10⁻⁵ t_(1/2) (h) 1.53± 0.20  2.69 ±  2.82 ± 2.66 ± 0.4 2.14 ± 0.34 0.42 0.35 The upper row ofdata at each pH provide the measured first order deactivation constant(k_(d) (1/s)) and lower row are the calculated half−lives (t_(1/2) (h)).

CT-PDMAEMA conjugates retained 80 to 90% of initial activity at pH 7 and40° C. after 6 h, whereas native CT retained only 50% of initialactivity (FIG. 8a ). The half-lives of all conjugates (182 to 237 h)were significantly longer than the native enzyme (7.2 h). Indeed, all ofthe conjugates retained around 70% of residual activity at pH 7 and 40°C. after incubation for 10 days, demonstrated in FIG. 9. At pH 7, all ofthe surface amine groups of the grafted PDMAEMA on the conjugates wouldhave been protonated. It was hypothesized that the considerable netadditional charge on each enzyme molecule (from the protonated subunitsin the conjugated polymers) would be sufficient to reduceprotein-protein interactions (FIG. 8a ). The stability of protonatedPDMAEMA-CT conjugates were significantly greater than that of PEGylatedCT. (Rodriguez-Martinez, J. A.; Rivera-Rivera, I.; Solá, R. J.;Griebenow, K. Biotechnol. Lett. 2009, 31, 883-887) As the pH increasedand net surface charges decreased, it was observed that CT and the PBPEconjugates had reduced half-lives. Since the average pK_(a) value ofPDMAEMA decreased with increasing molecular weight, (Dong, Z.; Wei, H.;Mao, J.; Wang, D.; Yang, M.; Bo, S.; Ji, X. Polymer 2012, 53,2074-2084.) longer grafted PDMAEMA conjugates would also tend to be morecollapsed and dehydrated at pH 8, thereby inhibiting autolysis throughsteric hindrance. Both native CT and the PBPE conjugates, when incubatedat pH 9, lost 70-80% of biocatalytic activity after 6 h (FIG. 8c ). AtpH 9, the amine groups of the PDMAEMA would be deprotonated anddehydrated. One could envisage that the now hydrophobic outer shell ofthe protein might have induced hydrophobic-hydrophobic interactions thatenhanced the degree of autolysis. At all pHs tested, the PBPE conjugateshad greater stability than native CT (Table 5).

Using PBPE, examples of dense PDMAEMA-CT conjugates with relativelynarrow molecular weight distributions have been synthesized. TheATRP-enabled PBPE approach of the present invention was used to tailorthe pH and temperature dependences of activity and stability of theenzyme chymotrypsin. The CT-PDMAEMA conjugates had higher relativeenzyme activities compared to native CT below pH 8. Indeed, theconjugates had a ten-fold higher enzyme activity than native enzyme atpH 5.0. The data demonstrated that points of inflection in thepH-activity profiles coincided with points at which the molecularconformation of the conjugate changed. It is observed that biocatalyticactivity was strongly influenced by the charge state, conformationalmorphology, and length of the grafted polymer, and that one couldrationally tailor these properties using pH and temperature as tunablevariables. The method of polymer-based protein engineering describedherein can be used to predictably alter the properties of a protein oreven to add a new functionality to an existing protein. Thewater-soluble, protein reactive ATRP initiator provides a key new toolin harnessing the potential of rationally combining biology and polymerscience.

A unique, water soluble ATRP initiator enabling polymer-based proteinengineering was designed and has been demonstrated and described inaccordance with several examples, which are intended to be illustrativein all aspects rather than restrictive. Thus, the present invention iscapable of many variations in detailed implementation, which may bederived from the description contained herein by a person of ordinaryskill in the art.

Experimental Series II Relating to Tailoring Enzyme Activity andStability

In the previous series of experiments, the ability and benefits ofbinding one type of polymer to multiple amine binding sites on a proteinsurface were demonstrated. In the second series of experiments, theability of the method of the present invention to bind more than onetype of polymer to a protein is shown. Because a purpose of polymerbased protein engineering is to modify the protein in a controlled andreliable manner to achieve, for example, a physiological benefit whenthe modified protein functions in vivo, polymers having a known responseto a biological parameter were chosen for the experiments.

Two polymers that show temperature responsiveness are poly(N-isopropylacrylamide) (pNIPAm) and poly [N,N′-dimethyl(methacryloylethyl) ammonium propane sulfonate] (pDMAPS), though theyrespond to temperature in sharply distinct ways. pNIPAm exhibits lowercritical solution temperature (LCST) behavior where above ˜32° C. thepolymer experiences a reversible change in conformation, increasing itshydrophobicity and becoming immiscible in water. The same reversiblechange is seen for pDMAPS, except that the polymer is immiscible belowthe upper critical solution temperature (UCST). The UCST of pDMAPS hasbeen shown to have strong dependence on polymer chain length andsolution ionic strength while the LCST of pNIPAm is less variable, butstill can be affected by several factors, such as degree of chainbranching and molecular weight.

A goal of this second experimental series was to controllably manipulatethe kinetics and stability of CT-pDMAPS and CT-pNIPAm bioconjugatesusing temperature as the trigger for a change in enzyme function. BothpNIPAm and pDMAPS were chosen in order to examine changes in relativeenzyme activity and stability at stimuli responsive temperatures bothabove and below room temperature. The contrasting temperature responsivebehavior of the UCST and LCST bioconjugates provided an attractiveapproach to examine how polymer chain collapse at varying temperaturesaffects enzyme bioactivity, stability, and substrate affinity.

The reaction is illustrated schematically as follows:

Reaction between the ATRP Initiator and Chymotrypsin

Synthesis of the ATRP initiator was carried out as described previously.Following synthesis, the ATRP initiator (469 mg, 1.4 mmol) and CT (1.0g, 0.04 mmol protein, 0.56 mmol—NH₂ group in lysine residues) weredissolved in sodium phosphate buffer (100 ml. of 0.1 M at pH 8.0). Thesolution was stirred at 4° C. for 3 h, then dialyzed against deionizedwater, using dialysis tubing with a molecular weight cut off of 15 kDa,for 24 h at 4° C. and then lyophilized.

Surface Initiated ATRP from CT-Initiator

To synthesize the CT-pDMAPS conjugates, the CT-Initiator complex (50 mg.0.024 mmol initiator) and DMAPS (335 mg. 1.2 mmol) were dissolved insodium phosphate buffer (20 mL, pH 6.0). In a separate flask, HMTETA (33μL, 0.12 mmol) was dissolved in deionized water (10 mL) and bubbled withArgon for 10 min. Cu(1) Br (17 mg. 0.12 mmol) was added to the HMTETAsolution and Argon was bubbled for an additional 50 min prior toaddition of the copper catalyst solution. The solution was then stirredfor 18 h at 4° C. Lastly, the solution was purified using dialysistubing with a molecular weight cut off of 25 kDa for 48 h againstdeionized water at 4° C., and the lyophilized.

For CT-pNIPAM synthesis, CT-Initiator conjugate (50 mg. 0.024 mmolinitiator) and NIPAm (271 mg. 2.4 mmol) were dissolved in deionizedwater (20 mL). In a separate flask, Me6TREN (32 μL. 0.12 mmol) wasdissolved in deionized water (10 mL) and bubbled with Argon for 10 min.Cu(I)Br (17 mg. 0.12 mmol) was added to the Me6TREN solution and Argonwas bubbled for an additional 10 min. The procedure for CT-pNIPAMsynthesis from this point forward was the same as described above forCT-pDMAPS synthesis.

Polymer Cleavage from CT Surface

Both PDMAPS and pNIPAm were cleaved from the surface of CT using acidhydrolysis. CT-pDMAPS conjugates were incubated (15 mg/mL) in 6 N HCl at110° C. under vacuum for 24 h. CT-pNIPAm (20 mg/mL) conjugates wereincubated in 4.5 N p-toluene sulfonic acid at 80° C. under vacuum for 72h. Following incubation, samples were isolated from CT using dialysistubing (MWCo 1 kDa) for 48 h. and then lyophilized. Lastly, polymermolecular weight was determined using GPC.

Gel Permeation Chromatography

Number and weight average molecular weights (M_(n) and M_(w)) and thepoly-dispersity index (M_(w)/M_(n)) were estimated by gel permeationchromatography (GPC). For pDMAPS, analysis was conducted on a Waters2695 Series with a data processor, using 80% 100 mM sodium phosphatebuffer (pH=9.0) 20% Acetonitrile with 0.01 volume % NaN₃ as an eluent ata flow rate 1 mL/min, with detection by a refractive index (RI)detector. Polystyrene sulfonate standards were used for calibration. ForpNIPAm, analysis was conducted using dimethylformamide (DMF) with 50 mMLiBr at a flow rate of 1 mL/min and 50° C., with detection by an RIdetector. Poly(ethylene oxide) standards were used for calibration anddiphenylethylene was used as a flow marker.

LCST/UCST Determination

CT-pDMAPS and CT-pNIPAm (2-3 mg polymer/mL each) were dissolved in 0.1 Mphosphate buffet (pH 8.0). CT-pNIPAm samples were heated from 20 to 35°C. and CT-pDMAPS samples were cooled from 30 to 5° C. at ±1° C./min. Theabsorbance at 490 nm was measured and LCST/UCST temperature wascalculated from the inflection point on the temperature versusabsorbance curves.

Dynamic Light Scattering

CT-pDMAPS (3 mg/mL) and CT-pNIPAm (0.5 mg/mL) samples were dissolved in0.1 M phosphate buffer (pH 8.0) and then filtered using a 0.45 μMcellulose filter. A Malvern Zetasizer nano-ZS was used to measurehydrodynamic radius (R_(h)). Each sample was measured in triplicate orgreater at each specified temperature.

CT and CT-Conjugate Biocatalytic Activity

N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide was used at a substrate forenzyme bioactivity assays. In a 1 mL cuvette, 0.1 M sodium phosphatebuffer (810-990 μL, pH 8.0, incubated at 25° C.), substrate (0-180 μL, 6mg/mL in DMSO (0-1.2×10⁻³M)), and enzyme (10 μL, 0.1 mg enzyme/mL 0.1 MpH 8.0 sodium phosphate buffer (4×10⁻⁸ M)) were mixed. The rate of thehydrolysis was determined by recording the increase in absorbance at 412nm for the first 30 s after mixing K_(M) and k_(cat) values werecalculated using EnzFitter software when plotting substrateconcentration versus initial rate.

Enzyme Stability

Native CT and CT conjugates (1 mg enzyme/mL) were dissolved in 0.1 Msodium phosphate buffer (pH 8.0) and incubated in a water bath at either25° C. or 40° C. At various time points, aliquots were removed anddiluted to 0.1 mg enzyme/mL using 0.1 M phosphate buffer (pH 8.0).Residual activity was calculated as the percentage of activity remainingrelative to the activity at time zero. Substrate (Suc-AAPF-pNA)concentration was kept constant at 288 μM for each sample and timepoints.

Results and Discussion

Reaction Between the ATRP Initiator and Chymotrypsin

In order to generate highly modified enzyme-polymer conjugates awater-soluble protein-reactive ATRP initiator was synthesized. Todetermine the efficiency of reaction between the enzyme (chymotrypsin)and the initiator, the increase in molecular weight was measured usingMALDI-TOF. On average, 12 ATRP initiating moieties were attached to eachCT molecule, through the reaction of an NHS-ester on the initiatormolecule with primary amine groups, either on surface accessible lysineresidues or the N-terminus. Consequently, for each CT molecule therewere 12 different sites from which polymer chains could be grown.Following conjugate synthesis, acid hydrolysis was used to cleave bothpDMAPS and pNIPAm from the surface of CT molecules for polymer molecularweight determination. Subsequent GPC analysis yielded number averagemolecular weight values (M_(n)) of 10 kDa for pDMAPS and 9 kDa forpNIPAm. From these M_(n) values, conjugate molecular weights werecalculated to be 148 kDa for CT-pDMAPS and 135 kDa for CT-pNIPAm. Nowthat it had been ensured that chymotrypsin could be modified through thehigh density attachment of thermo-responsive grown from polymers, thetemperature dependence of bioconjugate structure and function wasexplained.

Physical Properties of CT-pDMAPS and CT-pNIPAM Conjugates

The lower critical solution temperature (LCST) and upper criticalsolution temperature (UCST) behavior of the free polymers and CTbioconjugates were determined. The polymer component of thebioconjugates clearly responded to changes in temperature in the samemanner as the free polymer. For CT-pDMAPS, the UCST temperature was 13°C. which compares well with the UCST temperature for free pDMAPS polymer(12° C.). The LCST for the polymer component of CT-pNIPAm (˜29.5° C.)was only slightly lower than the LCST for free pNIPAm (30° C.). LCST andUCST transitions for the free polymer are representative of polymerchain collapse, but the thermodynamics governing these behaviors are notthe same. Phase transition during LCST is an entropy driven process,while UCST events are generally governed by changes in enthalpy. Foreach of the conjugates, the change from extended polymer to collapsedpolymer occurred slightly more rapidly than for free polymer (note thedifference in slope). It is possible that the protein-polymer interfacewas influencing the thermo-responsive behavior. Above a polymer's LCST,the polymer chains collapse and become insoluble in aqueous media,pushing water molecules to the outside of a newly formed hydrophobicpolymer shell. This behavior is reversible; thus, as the sample iscooled back to a temperature below the LCST, the polymer chainsrearrange, and are once again soluble in aqueous media. For a polymerthat exhibits UCST behavior, the polymer chains are extended andwater-soluble above the UCST, and collapsed/insoluble below the UCSTtemperature in aqueous media. When translating this temperaturesensitive behavior from free polymer to enzyme-polymer conjugates, itwas hypothesized that two different polymer conformations would belikely when temperature was varied above or below LCST/UCST temperature.Both above the LCST and below the UCST, the bioconjugates should have acollapsed, insoluble behavior. When below LCST and above UCST, anextended polymer component of the bioconjugate, with higher watersolubility compared to the collapsed state, should be in existence.

The cloud point curves for the bioconjugates were determined and showedthat there was phase separation and insolubility at the specific UCSTand LCST temperatures for each of the CT-polymer conjugates. The impactof temperature on bioconjugate size was examined. The hydrodynamicradius (R_(h)) for the CT-pDMAPS and CT-pNIPAm conjugates, measured byDynamic Light Scattering (DLS) at temperatures of interest near the LCSTand UCST, was temperature-dependent. Owing to the longer polymer chain,CT-pNIPAm conjugates had a larger extended state R_(h) of ˜8 nm comparedwith ˜6.5 nm for CT-pDMAPS. The hydrodynamic radius decreased above theLCST for CT-pNIPAm and below the UCST for CT-pDMAPS as the polymerscollapsed and became hydrophobic. We hypothesized that as the polymerscollapsed, they more fully covered the surface area of the proteinrather than extending outward. The specific phase separation behaviorfor each conjugate exhibited in the cloud point curves was conserved inthe R_(h) measurements. The CT-pDMAPS UCST transition encompassed alarger temperature range when compared with CT-pNIPAm. For CT-pDMAPS, agradual decrease in R_(h) plateaued at ˜3.8 nm around 15° C. Incomparison, CT-pNIPAm conjugates showed a more rapid phase transitionwith the R_(h) quickly decreasing from 30 to 31° C. The quick formationof hydrophobic aggregates (high R_(h)) for each CT-polymer conjugate atextreme temperatures prevented the examination of the R_(h) attemperatures further away from the UCST and LCST. We next completed anexhaustive analysis of the chymotrypsin bio-conjugate activity andspecificity as a function of temperature.

Bioconjugate Activity

Overall enzyme activity was retained during cycling of CT-pNIPAm andCT-pDMAPS conjugates above and below their respective LCST and UCSTtemperatures. As the polymers in the conjugate switched between acollapsed and extended state, no large decrease in conjugate residualactivity was observed.

The kinetic constants (k_(cat)) and K_(M)) were determined at threedifferent temperatures (5° C., 25° C., and 40° C.) for each of theconjugates and native CT, using Suc-AAPF-pNA as the model substrate.These temperatures were chosen so as to observe enzyme function aboveand below the UCST and LCST that we determined for the bioconjugates. At25° C., both CT-pDMAPS and CT-pNIPAm polymers were in their polymerchain extended state.

At 25° C., CT-pDMAPS conjugates showed similar k_(cat)/K_(M) values tonative CT, while CT-pNIPAm conjugates showed slightly lowerk_(cat)/K_(M) values (Table 6 shown in FIG. 26). In addition,CT-Initiator conjugates showed increased k_(cat)/K_(M) values at eachtemperature. The ATRP initiator molecule was covalently coupled to theCT surface through the amine side group on lysine residues. Due to thisattachment technique, CT surface charge was modified after initiatorimmobilization, and this modification is believed to be responsible forthe increase in bioactivity seen for the CT-Initiator conjugate at allthree temperatures.

Several interesting trends were observed when closely examining thetemperature dependence of k_(cat) and K_(M) for the CT-pDMAPS andCT-pNIPAm conjugates. for each temperature, the relative k_(cat) ratiofor CT-pDMAPS stayed constant at ˜0.75. At all three temperatures, forCT-pDMAPS conjugates, K_(M) was lower when compared with native CT,meaning there was higher substrate affinity with the CT-pDMAPS conjugatethan native CT. It has been hypothesized that reduced K_(M) values forCT-zwitterionic polymer conjugates resulted from the interaction of themodel substrate with pDMAPS polymer. Taking a similar approach, it canbe hypothesized that the model hydrophobic substrate for CT used in thisstudy interacted with the hydrophilic pDMAPS polymer surrounding CT,increasing the local concentration of the substrate near the hydrophobicsubstrate binding pocket, thereby lowering K_(M) for CT-pDMAPS. As shownin the relative K_(M) values, this higher affinity was seen at eachtemperature, but was reduced, perhaps by the collapsed nature of pDMAPS,below the UCST. At 5° C., the relative K_(M) value for CT-pDMAPS washigher when compared to relative K_(M) values at 25° C. and 40° C. Attemperatures below the UCST of CT-pDMAPS (13° C.), the polymer was inits collapsed state. It is not unreasonable to presume that once pDMAPSwas in a collapsed state it would have restricted the access of thesubstrate to the active site via steric hindrance.

At 40° C., a sharp decrease in CT-pNIPAm bioactivity was seen with arelative k_(cat)/K_(M) value of 0.12. At this temperature pNIPAm was inits collapsed, hydrophobic state, and K_(M) likely increased due tosteric hindrance. In addition, since pNIPAm is more hydrophobic thanpDMAPS, pNIPAm would have a stronger association with the hydrophobicmodel substrate. It was likely, then, that the long and dense pNIPAmmolecules could partition the substrate in the polymer phase, therebyincreasing K_(M). The interaction of pNIPAm with the substrate, whichcan be seen from the increase in K_(M) at 25° C., was also exhibited atother temperatures. For CT-pNIPAm conjugates, relative k_(cat) valueswere similar at both 5° C. and 25° C., and only slightly lower thannative CT. At 40° C., the first order rate constant (k_(cat)) was muchlower for CT-pNIPAm conjugates when compared to native CT, and it washypothesized that k_(cat) decreased because of a decrease in wateravailability at the active site. CT catalyzes peptide bond hydrolysisthrough a charge stabilizing amino acid triad, and consequently, wateris needed for the reaction to occur. As pNIPAm polymer chainssurrounding CT collapse above the LCST, the polymer would be expected toalter the mobility of enzyme bound water molecules. Changes in watermobility at the CT-pNIPAm active site above pNIPAm LCST would beobserved in a reduced k_(cat), as observed. These two factors arebelieved to have caused a lower bioactivity for CT-pNIPAm conjugates at40° C.

The impact of the polymer-based protein engineering on enzyme stabilitywas assessed.

TABLE 7 Temperature dependence of first order inactivation rateconstants and half-lives for chymotrypsin and polymer-based proteinengineered chymotrypsin. 25° C. 40° C. Sample k_(inact) (days−1) t_(1/2)(days) k_(inact) (days⁻¹) t_(1/2) (days) Native CT 0.13 ± 1.2 × 10⁻²5.43 ± 0.51 6.61 ± 0.47 0.10 ± 7.5 × 10⁻³ CT-Initiator 0.26 ± 2.0 × 10⁻²2.67 ± 0.21 23.8 ± 3.1  0.03 ± 3.8 × 10⁻³ CT-pDMAPS 0.05 ± 8.3 × 10⁻³14.2 ± 2.39 1.69 ± 0.16 0.41 ± 3.9 × 10⁻² CT-pNIPAm 0.01 ± 3.4 × 10⁻³61.2 ± 18.7 1.04 ± 0.14 0.66 ± 8.8 × 10⁻² Stabilities of native CT andCT conjugates were determined by incubating 1 mg enzyme/mL. Theinactivation constants (k_(inact)) and half-lives (t_(1/2)) werecalculated by fitting a first order decay to the data.Polymer-Based Protein Engineering of Enzyme Stability

A first order inactivation model was used to examine the irreversiblethermal inactivation of native CT and the bioconjugates at both 25° C.and 40° C. the CT-pNIPAm and CT-pDMAPS conjugates showed dramaticallyenhanced stability compared to native CT and initiator modified CT.While CT-conjugate stability was higher at both temperatures, thedeactivation mechanisms at these temperatures are likely to differ. At25° C., CT inactivation is due mostly to autolysis whereas at 40° C.both protein structure denaturation and autolysis contribute to theirreversible inactivation of CT. In addition, the stabilizationmechanisms for pNIPAm and pDMAPS were likely different. It was expectedthat pNIPAm would dampen the structural dynamics of CT therebypreventing structural unfolding in a manner similar to that observedafter protein PEGylation. In contrast, pDMAPS likely formed chargeinteractions between the polymer and protein given its zwitterionicstructure thereby stabilizing the protein. While different, bothmechanisms dramatically increased stability of CT-polymer conjugates atboth 25° C. and 40° C. (Table 7). The half-lives of the bioconjugateswere orders of magnitude greater than the native chymotrypsin.

In addition to higher general conjugate stability than native CTstability, at both experimental temperatures (25° C. and 40° C.), thestability of CT-pNIPAm conjugates was higher compared to CT-pDMAPSconjugates. At 25° C., both CT-pNIPAm and CT-pDMAPS polymers were intheir extended state. The higher stability of CT-pNIPAm was attributedto the lower activity values seen in Table 6 in FIG. 26. Since autolysiswas the main contributor to CT denaturation at 25° C., the loweractivity values seen as this temperature corresponded to a higherstability. At 40° C., CT-pNIPAm was in its collapsed state, which likelycaused a decrease in autolysis by blocking CT molecules access to theactive site. The effect of collapsed versus expanded state for thepolymer on the tertiary structure of the protein is not known at thistime, but is a potential topic for future studies. At 40° C., CT-pDMAPSwas in its extended state, and still provided increased stabilitycompared to native CT through steric hindrances and structuralstabilization, but to a lower degree than CT-pNIPAm conjugates.

Conclusion

Polymer-based protein engineering alters in a controlled manner thetemperature dependence of relative enzyme activity, stability, andsubstrate affinity. LCST behavior in pNIPAm and UCST behavior in pDMAPSpolymers were conserved in the enzyme-polymer bioconjugates grown fromthe surface of chymotrypsin. In addition, enzyme bioactivity wasconserved when activity assays were conducted at temperatures where theconjugates were in both their extended and collapsed states.Interactions between the model substrate and the polymer surrounding theprotein core influenced changes in relative substrate affinity (K_(M)),although pDMAPS and pNIPAm showed opposing behavior. Relative substrateaffinity was increased in CT-pDMAPS conjugates (lower K_(M)), butdecreased (higher K_(M)) in CT-pNIPAm conjugates. When above the LCSTand below the UCST (polymer collapsed state), relative activity of theconjugates was maintained, though slightly reduced, while increasing CTstability to autolysis and denaturation. CT-conjugate stability was alsohigher compared to native CT at 25° C., where the polymer is in itsextended, non-temperature responsive conformation. In summary, theseresults show that water-soluble protein-reactive ATRP initiator could beused as the foundation of a polymer-based protein engineering strategydesigned to tailor the temperature dependence of enzyme stability,activity and specificity.

Experimental Series III Relating to Rational Tailoring of Substrate andInhibitor Affinity Via ATRP Polymer-Based Protein Engineering

The various embodiments of the polymer-based protein engineering methodsdescribed herein add significantly to the rational design of polymers,whether synthetic or biological, grown from the surface of proteins tospecifically alter protein structure and function. Polymer-based proteinengineering focuses on rational tailoring of polymer choice based ontargeted benefits of polymer conjugation, rather than the chemistry usedto yield such bioconjugates. Polymer-based protein engineering is asimpler synthetic approach than those heretofore used, similar inoutcomes to protein glycosylation, a natural post-translationalmodification that assists with correct protein folding and helpsregulate cellular functions, such as cell-cell communication, that aredependent upon protein signaling. (see Elmouelhi, N. Y., K. J. InBiotechnol Bioeng; Flynne, W. G., Ed.; Nova Science Publishers, Inc.:New York, 2008, p 37.) Protein function can be altered by polymer-basedprotein engineering using synthetic or biologically inspired monomersmuch like glycosylation. With polymer-based protein engineering, some ofthe advantages that come with glycosylation, such as improved stability,can be imparted onto the protein without often complicated biologicaltechniques.

As described extensively herein, protein-polymer conjugates with highdensity polymers around the protein core can be synthesized using the“grafting from” approach.

In this series of experiments, a massive reversal of chymotrypsin (CT)surface charge using polymer-based protein engineering withpoly(quaternary ammonium) (pQA) is described. pQA is a cationic polymerthat has commonly been used for antibacterial applications. (Chen, C.Z.; Beck-Tan, N. C.; Dhurjati, P.; van Dyk, T. K.; LaRossa, R. A.;Cooper, S. L. Biomacromolecules 2000, 1, 473; Huang, J.; Koepsel, R. R.;Murata, H.; Wu, W.; Lee, S. B.; Kowalewski, T.; Russell, A. J.;Matyjaszewski, K. Langmuir 2008, 24, 6785) Other cationic syntheticpolymers have seen wide use in delivery of both RNA (Sioud, M.;Sorensen, D. R. Biochemical and Biophysical Research Communications2003, 312, 1220; Averick, S. E.; Paredes, E.; Irastorza, A.; Shrivats,A. R.; Srinivasan, A.; Siegwart, D. J.; Magenau, A. J.; Cho, H. Y.; Hsu,E.; Averick, A. A.; Kim, J.; Liu, S.; Hollinger, J. O.; Das, S. R.;Matyjaszewski, K. Biomacromolecules 2012, 13, 3445.) and DNA (Benns, J.M.; Choi, J.-S.; Mahato, R. I.; Park, J.-S.; Kim, S. W. BioconjugateChem 2000, 11, 637.) nucleotide based therapies to enable transport ofdrugs across the cell membrane. Modification of enzyme surface charge bysite directed mutagenesis or synthetic chemistry has also been shown tocause dramatic effects on protein function. Specifically, modifyingprotein surface charge has been shown to influence the stability andactivity profiles of enzymes in non-aqueous solvents such as ionicliquids (Nordwald, E. M.; Kaar, J. L. Biotechnol Bioeng 2013, 110, 2352)as well as shifting the pH-profile of enzyme activity (Russell, A. J.;Fersht, A. R. Nature 1987, 328, 496; Sandanaraj, B. S.; Vutukuri, D. R.;Simard, J. M.; Klaikherd, A.; Hong, R.; Rotello, V. M.; Thayumanavan, S.J Am Chem Soc 2005, 127, 10693).

Herein, “grafting from” ATRP to form a high density cationic shellaround the chymotrypsin core is described. As stated above, exogenouschymotrypsin dosing could be used to treat pancreatic exocrinedeficiency, but low stability to stomach acid degradation of unmodifiedchymotrypsin would likely require high dosing. It was hypothesized thatthe high density cationic pQA shell surrounding chymotrypsin woulddramatically increase stability, shift the pH profile of chymotrypsinactivity, and influence inhibitor binding. Four different molecularweight chymotrypsin-pQA conjugates were synthesized to study the effectof PBPE surface charge modification on enzyme kinetics, stability, andinhibitor affinity.

Synthesis of 2-(dimethylethylammonium)ethyl methacrylate (QA Monomer)

2-(dimethyamino)ethyl methacrylate (21.4 mL, 127 mmol) and bromoethane(11.4 mL, 153 mmol) were added in acetonitrile (80 mL). The mixture wasstirred at 35° C. overnight. After Diethyl ether (200 mL) was added,crystalized QA monomer was filtered off, washed with diethyl ether, anddried in vacuo; yield 33.7 g (99%), mp 112-114° C. ¹H NMR (300 MHz,DMSO-d₆) δ 1.26 (t, 2H, J=7.2 Hz, N⁺ (CH₃)₂CH₂CH₃), 1.90 (s, 3H, ═CCH₃),3.12 (s, 6H, N⁺ (CH₃)₂CH₂CH₃), 3.49 (q, 2H, J=7.2 Hz, N+(CH₃)₂CH₂CH₃),3.74 (t, 2H, J=4.5 Hz, OCH₂CH₂N⁺), 4.51 (t, 2H, OCH₂CH₂N⁺), 5.75 (s, 1H,CH═), and 6.08 (s, 1H, CH═) ppm. IR (KBr) 3447, 2986, 1718, 1635, 1457,1320, 1299, 1168, 1014, 958, 816, 646, 603, and 554 cm⁻¹.

Dynamic Light Scattering (DLS).

The DLS data were collected on a Malvern Zetasizer nano-ZS, which waslocated in the Department of Chemistry, Carnegie Mellon University,Pittsburgh, USA. The concentration of the sample solution was kept at1.0 mg/mL. Hydrodynamic diameters (D_(h)) of native CT and conjugateswere measured three times (12 runs/measurement) in 100 mM sodiumphosphate buffer (pH 7.0) at 25° C.

“Grafting from” ATRP of QA Monomer from the CT-ATRP Initiator Conjugate.

A solution of QA monomer (284 mg, 1.07 mmol (CT-pQA₂₅); 567 mg, 2.13mmol (CT-pQA₅₀); 1.13 g, 4.26 mmol (CT-pQA₁₀₀); 2.27 g, 8.52 mmol(CT-pQA₂₀₀) and CT-initiator conjugate (α-Chymotrypsin-NHS ATRPinitiator) (100 mg, 0.043 mmol of initiator groups) in de-ionized water(30 mL) was sealed and bubbled with Argon in an ice bath for 50 min.Deoxygenated catalyst solutions of HMTETA (24 μL, 0.2 mmol) and Cu(I)Br(13 mg, 0.2 mmol) in de-ionized water (10 mL) was added to theconjugation reactor under Argon bubbling. The mixture was sealed andstirred in a refrigerator for 4 h. CT-pQA conjugates were isolated bydialysis with a 25-kDa molecular weight cut-off dialysis tube inde-ionized water in a refrigerator for 24 h, and then lyophilized.

Cleavage of the Grafted PQA from the Conjugate.

Chymotrypsin-pQA conjugates (10 to 20 mg) and 6 N HCl aq. (2 to 3 mL)were placed in separate hydrolysis tubes. After three freeze-pump-thawcycles, hydrolysis was performed at 110° C. for 24 h in vacuum. Thecleaved polymer was isolated using a 1 kDa molecular weight cut offdialysis tubing in de-ionized water and then lyophilized. The molecularweight of the cleaved polymer was measured by GPC.

Determination of molecular weight of the prepared conjugates. Molecularweights of the prepared CT-pQA conjugates were calculated from estimatedmolecular weight of cleaved PQA from the conjugate. BCA and absorptionassays were also carried out to determine molecular weight of theconjugates. Detailed procedures for BCA and absorption molecular weightcalculations are provided in the experimental series above.

Enzyme Activity

N-Succinyl-Ala-Ala-Pro-Phe p-nitroanilide (0 to 50 μL of 9.60 mM inDMSO) was added to sodium phosphate buffer (990 to 940 μL of 100 mM, pH5-10). Native CT or conjugates solution (10 μL of 4.0 μM) was added tothe substrate solution. The initial rate of hydrolysis of the peptidesubstrate was monitored by recording the increase in absorption at 412nm using a UV-VIS spectrometer. The Michaelis-Menten parameters(k_(cat), K_(M) and k_(cat)/K_(M)) were determined by non-linear curvefitting (using the well known Michaelis-Menten kinetics equation,V=V_(max)[S]/K_(M)+[S], k_(cat)=V_(max)[E] where [E] is enzymeconcentration, [S] is substrate concentration, and V is initial rate ofthe reaction) of plots of initial rate versus substrate concentrationusing the Enzfitter software. Table 8 below provides theMichaelis-Menten parameters of hydrolysis of Suc-AAPF-pNA from pH 5-10.

TABLE 8 pH 5.0 pH 6.0 pH 7.0 pH 8.0 pH 9.0 pH 10.0 native k_(cat)  2.85± 14.80 ± 26.81 ± 32.00 ± 33.30 ± 36.62 ± 0.21 1.07 0.80 1.18 1.31 3.11K_(M) 184.0 ± 171.1 ±  94.3 ±  82.6 ± 122.7 ± 336.3 ± 31.1 28.4 11.1 9.312.7 52.7 k_(cat)/ 0.016 ± 0.086 ± 0.285 ± 0.387 ± 0.272 ± 0.109 ± K_(M)0.003 0.016 0.034 0.045 0.029 0.019 C1 k_(cat)  5.06 ± 10.87 ± 14.49 ± 17.5 ± 13.26 ±  9.96 ± 0.12 0.24 0.17 0.34 0.61 0.30 K_(M) 68.5 ± 5.154.1 ± 4.1 32.0 ± 1.6  34.4 ± 2.8 46.61 ± 72.6 ± 8.05 6.8 k_(cat)/ 0.074± 0.201 ± 0.453 ± 0.509 ± 0.285 ± 0.137 ± K_(M) 0.006 0.016 0.024 0.1070.051 0.013 C2 k_(cat)  5.50 ± 13.57 ± 16.57 ± 20.95 ± 15.21 ±  7.54 ±0.16 0.27 0.18 0.30 0.60 0.16 K_(M) 70.8 ± 6.5 56.0 ± 3.8 31.8 ± 1.6 36.0 ± 54.0 ± 7.5  51.2 ± 2.1 4.0 k_(cat)/ 0.078 ± 0.242 ± 0.521 ±0.582 ± 0.282 ± 0.147 ± K_(M) 0.008 0.017 0.027 0.035 0.041 0.012 C3k_(cat)  4.42 ±  9.24 ± 13.06 ± 17.22 ± 15.77 ± 12.96 ± 0.10 0.29 0.220.64 .78 0.38 K_(M) 55.2 ± 4.3 39.9 ± 4.9 34.1 ± 2.4 43.6 ± 6.2  62.7 ±10.3 88.4 ± 7.6 k_(cat)/ 0.080 ± 0.231 ± 0.383 ± 0.395 ± 0.252 ± 0.147 ±K_(M) .007 0.030 0.028 0.058 0.043 0.013 C4 k_(cat)  4.73 ± 0.12 10.85 ±15.59 ± 21.73 ± 18.89 ±  9.86 ± 0.29 0.27 0.40 1.17 0.24 K_(M) 59.4 ±5.2 49.8 ± 4.9 29.3 ± 2.4  36.2 ±  62.5 ±  57.6 ± 2.7 12.8 4.9 k_(cat)/0.079 ± 0.219 ± 0.521 ± 0.601 ± 0.302 ± 0.171 ± K_(M) 0.007 0.023 0.0430.047 0.065 0.015 Units are as follows: k_(cat), sec⁻¹; K_(M), μM;k_(cat)/K_(M), sec⁻¹ · μM⁻¹.

Enzyme activity (k_(cat)), specificity (K_(M)), and productivity(k_(cat)/K_(M)) values were determined for each of the conjugates andnative chymotrypsin at 25° C. in 0.1 M phosphate buffer (pH 5-10).Values were determined by monitoring the enzyme catalyzed hydrolysis ofSuc-AAPF-pNA. Enzyme concentration for each of the reactions was 39 nM,with substrate concentrations ranging from 0-700 μM. The reaction wasmonitored for the first 30 seconds after mixing enzyme and substrate.

Stability

Native CT and conjugates (1.5 to 2.0 mL, 4.0 μM) were incubated in 100mM sodium phosphate buffer (pH 7.0) at 50 or 60° C., and an artificialgastric acid solution (167 mM HCl aq. pH 1.5) at 37° C. Aliquots (50 μL)were removed and kept at 0° C. before measuring residual activity. Theresidual activity was calculated as a ratio of initial rates ofhydrolysis reaction at given incubation time over the initial activityfor each specific sample.

Inhibitor Binding Study.

Enzyme inhibition was assayed by measuring the hydrolytic activities ofsamples which contained a fixed concentration of protease and varyingamounts of inhibitor, including a blank with no inhibitor. Inhibitorsand peptide substrate were simultaneously added to the native andconjugate solutions immediately before measuring initial rates. Theinitial rate of hydrolysis of the peptide substrate was monitored byrecording the increase in absorption at 412 nm. To determinedissociation constant of the enzyme-inhibitor complexes (Ki),V_(max app) and K_(M app) were determined by non-linear curve fitting(equation for Michaelis-Menten parameters) of plots of initial rateversus substrate concentration using the Enzfitter software. Thedissociation constants were calculated from secondary plot ofK_(M app)/V_(max app) versus initial inhibitor concentration.

Results and Discussion

Polymer-Based Protein Engineering of CT with a Positively ChargedPolymer

In order to test the hypothesis that a massive reversal of surfacecharge would affect protein stability, inhibitor binding, and the pHprofile of enzyme activity, the surface charge of chymotrypsin wasmodified by growing cationic pQA from multiple sites on the surface ofchymotrypsin. Before polymerization, a water soluble, brominefunctionalized ATRP initiator was reacted with lysines on the surface ofchymotrypsin and the N-terminus using basic NHS chemistry readilyavailable in the literature and well known in the art. Afterimmobilization, MALDI-TOF-MS was used to determine that there were 12initiating sites per molecule of chymotrypsin. Next, the reactionconditions were varied as described above to synthesize four distinctchymotrypsin-pQA conjugates (Scheme II).

To measure molecular weight of the polymers, acid hydrolysis was used tocleave the polymers from chymotrypsin followed by molecular weightanalysis with size exclusion chromatography. From these measurements,the average degree of polymerization (DP) was calculated for thepolymers in each conjugate, which is the motivation for the nomenclatureused for each of the conjugates (CT-pQA₂₇, CT-pQA₅₄, CT-pQA₁₀₈,CT-pQA₁₉₈)). The DP of the polymeric quaternary ammonium was easilycontrolled by the initial molar concentration of QA monomer. The data isshown in Table 9.

TABLE 9 Impact of polymerization reaction conditions on molecular weightand size of cationic shell encapsulated chymotrypsin polymerizationCleaved Molecular weight of conjugate (kDa) Condition¹ Yield² Polymer³cleaved D_(h) ⁶ sample [I]₀:[M]₀ (%) M_(n) (M_(w)/M_(n)) polymer⁴ BCA⁵Absorption⁵ (nm) CT-pQA₂₈ 1:25  58  7,400 (1.51) 114.3 96.6 96.5 13.7 ±3.3 CT-pQA₅₄ 1:50  75 14,500 (1.86) 199.5 150.5 164.3 14.7 ± 4.5CT-pQA₁₀₈ 1:100 93 28,600 (2.04) 368.7 289.2 286.1 19.2 ± 2.4 CT-pQA₁₉₈1:200 88 52,600 (1.95) 636.7 578.5 575.4 28.6 ± 6.1 ¹12 initiator unitsper CT-initiator conjugate, [I]0:[Cu(I)Br]0:[HMTETA]0 = 1:2:2, DI water,4° C., 4 h. ²Yield (%) is the total weight of lyophilized CT-pQAconjugate/total weight of loaded CT-initiator and QA monomer × 100.³Grafted pQA was cleaved by vacuum hydrolysis method using 6N HCl at110° C, 24 h. Cleaved polymer was isolated by dialysis (Mwco 1,000).Molecular weight of cleaved polymer was estimated by GPC. ⁴Molecularweight of conjugate is reported as Mn of cleaved polymer × 12 + 28,000(molecular weight of CT-initiator). ⁵Methods for molecular weightdetermined by BCA and absorption methods are provided in SupplementaryMaterials section. ⁶Hydrodynamic diameter of the chymotrypsin-pQAconjugate was measured using dynamic light scattering with sample conc.1.0 mg/mL in 100 mM sodium phosphate (pH 7.0) at 25° C.

For the longest chain conjugate (CT-pQA₁₉₈), the conjugate molecularweight was increased over twenty fold from native chymotrypsin. Thehydrodynamic diameters (D_(h)) of the conjugates were determined usingdynamic light scattering (DLS) in 100 mM sodium phosphate buffer (pH 8).As expected, the D_(h) of chymotrypsin-pQA increased with the length ofthe grown pQA, and CT-pQA₁₉₈ D_(h) was increased ˜6 fold over nativechymotrypsin. PBPE of chymotrypsin using ATRP provided us with a methodto rationally control the molecular weight and size of chymotrypsin-pQAconjugates. The molecular weight of the conjugates described in thisseries of experiments are much higher than that described hereinabovefor the first series of chymotrypsin polymer conjugates. The longestchain conjugate (polymer molecular weight 53 kDa) is double the lengthof the longest grown pH-responsive polymers, and 1.5 times the length ofthe longest temperature-responsive grown block copolymer.

Impact of PBPE on Enzyme Bioactivity

Following synthesis and physical characterization, the effect ofpositively charged polymer shells on bioactivity and specificity wasexamined. Chymotrypsin-pQA bioactivity was determined for each conjugateby monitoring the rate of enzyme catalyzed hydrolysis ofN-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide from pH 5-10 at 25° C.Conjugation of the positively charged pQA to chymotrypsin had an effecton both KM and k_(cat) at all pH's (FIG. 15). However, the activity ofchymotrypsin-pQA conjugates were not dependent upon molecular weight ofthe conjugated polymer. (FIG. 15A).

Between pH 7 and 10 the relative k_(cat) for each conjugate was abouthalf of that for native chymotrypsin. The turnover number for enzymesconjugated with a large number and density of polymers are oftendecreased, likely due to reduced structural dynamics of the protein (seeRodriguez-Martínez, J. A.; Solá, R. J.; Castillo, B.; Cintrón-Colón, H.R.; Rivera-Rivera, I.; Barletta, G.; Griebenow, K. Biotechnol Bioeng2008, 101, 1142.), so this result was not surprising. At lower pH's (pH5 and 6), the activity ratios for the chymotrypsin-pQA conjugates ofeach molecular weight were increased compared to kcat values at higherpH's. Chymotrypsin catalytic activity is dependent upon a chargestabilizing amino acid triad consisting of Ser195, Asp102, and His57. Inthis triad, a negative charge on Asp102 is required for enzyme catalyzedhydrolysis to proceed. In native chymotrypsin, the carboxyl group ofAsp102 would be protonated at low pH, and the charge stabilizing effectwould be expected to be absent, greatly reducing enzyme activity. Forchymotrypsin-pQA, the hypothesis was that the positive charge densityimparted by pQA polymer shell stabilized the negative charge on Asp102,which shifted the pH profile for chymotrypsin-pQA conjugates.

It was observed that relative K_(M)'s were not dependent upon chainlength (as for k_(cat)), and were also independent of pH (FIG. 16B).K_(M)'s for each of the chymotrypsin-pQA conjugates were greatlydecreased at each pH, indicating higher substrate affinity compared tonative chymotrypsin. At each pH (5-10) relative K_(M) values werebetween ˜0.2 and ˜0.5. The lower K_(M) values for chymotrypsin-pQA,which have been seen before with charged polymers conjugated tochymotrypsin, were likely due to a partitioning effect between thesubstrate and the hydrophilic polymer shell around chymotrypsin. It hasbeen suggested that the hydrophobic substrate exhibited a preferentialaccumulation in the hydrophobic active site pocket of chymotrypsinrather than the pQA shell. (see Keefe, A. J.; Jiang, S. Y. Nat Chem2012, 4, 60) The data produced in these experiments tended to supportthis hypothesis, although it was interesting to observe that thesubstrate readily crossed the shell layer since activity was observed.

Enzyme productivity (k_(cat)/K_(M)) for each of the chymotrypsin-pQAconjugates was, of course, higher than native chymotrypsin from pH 5 to10 (FIG. 15C). Relative productivity values were highest at pH 5 (5 foldincrease) and 6 (2-3 fold increase), where the increased enzyme activity(k_(cat)) and higher substrate affinity (K_(M)) for each molecularweight conjugate improved the performance over native chymotrypsin.Chymotrypsin-pQA conjugates also showed 1-2 fold increases in enzymeproductivity at pH values from 7-10. At these higher pH values, theincrease in enzyme productivity was due mainly to the lower K_(M) valuesas discussed above.

Conjugate Stability at Extremes of Temperature and pH

Next, the stability of native chymotrypsin and chymotrypsin-pQA wereexamined at extremes of temperature and pH. Chymotrypsin-pQA conjugatesof each molecular weight were more stable than native chymotrypsin atboth 50° C. (FIG. 16A) and 60° C. (FIG. 16B) in 0.1 M sodium phosphatebuffer (pH 8.0). Unlike bioactivity and substrate affinity, the degreeof increase in stability for chymotrypsin-pQA conjugates was dependenton polymer molecular weight.

At 50° C., native chymotrypsin lost all activity after 2 h, while allchymotrypsin-pQA conjugates maintained at least 40% of initial activityafter 8 h of incubation. At 60° C., where native chymotrypsin lost allactivity in 30 min, similar increases in chymotrypsin-pQA stability wereobserved. CT-pQA₁₉₈ maintained 35% of initial activity after 8 hours. Atthese extreme temperatures and at the optimum pH for CT (pH 8), it isbelieved that two factors influenced irreversible thermo-inactivation ofthe enzyme. First, chymotrypsin molecules unfolded from their nativestructure which led to inactivation. Second, after unfolding, access forpeptide bond cleavage by other CT molecules (autolysis) was increased.It is believed that each of these mechanisms contributed to CTinactivation at high temperature, and that the growth of pQA from thesurface of the enzyme at high density stabilized the enzyme byinhibiting both of these processes. First, it was possible that cationicammonium ions in the backbone of pQA could have stabilized the tertiarystructure of CT similarly to ion salt stabilization in solutionfollowing the Hoffmeister series. (see Baldwin, R. L. Biophys J 1996,71, 2056) Second, it has been observed previously that steric hindranceof the polymers around the CT core restricted access for CT molecules toperform autolysis. At both 50° C. and 60° C., the degree ofstabilization was polymer chain length dependent. Enzymes are heldtogether by thousands of interactions, but the balance of forces thatcreate a stable structure is delicate. It has been known for a long timethat multipoint cross-linking and enzyme crystallization candramatically stabilize enzymes. A covalently coupled layer of highdensity polymer that cannot collapse would be expected to preventunfolding by adding hundreds of polymer-polymer stabilizing forces tothe network that retains the structure of the protein.

Chymotrypsin-pQA conjugates of each chain length were also remarkablystable in 167 mM HCl (pH 1) at 37° C. (FIG. 17). Native chymotrypsinlost all activity after 3 hours, but each chymotrypsin-pQA conjugatemaintained at least 40% of activity after 8 hours. Chymotrypsin-pQAconjugates were still active up to 99 hours after incubation at this lowpH. At pH 1, stabilization of chymotrypsin by the pQA shell must haveresulted from structural stabilization since autolysis cannot contributeat pH 1. It has been shown herein that a charged block copolymer enzymeshell dramatically stabilized chymotrypsin at low pH by inhibitingprotein unfolding. As was seen with increased stability at elevatedtemperature, the stability profiles for chymotrypsin-pQA conjugates weredependent on polymer chain length. Chymotrypsin-pQA₂₇ showed the loweststability of the conjugates, while chymotrypsin-pQA₁₉₈ showed thegreatest increase in stability. Interestingly, stability profiles of theconjugates at low pH showed a consistent trend with polymer chainlength. Conversely, stability for chymotrypsin-pQA conjugates atelevated temperature showed a biphasic behavior where chymotrypsin-pQA₂₇and chymotrypsin-pQA₅₄ had similar, but lower stability andchymotrypsin-pQA₁₀₈ and chymotrypsin-pQA₁₉₈ had similar, increasedstability. Since chymotrypsin is not active at low pH and autolysiscould not contribute to inactivation, it is likely that the polymer onlystabilized chymotrypsin structurally through charge effects.Classically, ions were thought to stabilize proteins based on theirability to create a more or less ordered bulk water structure (LoNostro, P.; Ninham, B. W. Chem Rev 2012, 112, 2286). More recently, itis believed that direct interactions between ions and surface residuesin the protein play a more important role in determining proteinstability (Paterova, J.; Rembert, K. B.; Heyda, J.; Kurra, Y.; Okur, H.I.; Liu, W. S. R.; Hilty, C.; Cremer, P. S.; Jungwirth, P. J Phys Chem B2013, 117, 8150.; Zhang, Y.; Cremer, P. S. Current Opinion in ChemicalBiology 2006, 10, 658.). Still, the end result of these interactions isa “salting out” effect, where tertiary structure is stabilized bystrengthened hydrophobic interactions in the protein. Within thecationic pQA shell, the local concentration of these stabilizing ionswas high which contributed to the increased stability ofchymotrypsin-pQA conjugates at both high temperature and low pH.

Rational Tailoring of Enzyme-Inhibitor Binding

Next, the effect of the dense cationic pQA shell around the surface ofCT towards inhibitor binding of positively and negatively chargedinhibitors was examined. Two protein inhibitors, aprotinin (AP) andBowman-Birk trypsin-chymotrypsin inhibitor from glycine max (GM), werechosen due to their contrasting surface charges. GM with a pI (4-4.3)41below the tested pH (8) had negative surface charge, and AP, which has apI of 10.542, had a positive surface charge. The dense cationicmolecular shell should have predictable effects on the ability of theinhibitors to bind to the enzyme. AP was not as effective as aninhibitor for chymotrypsin-pQA conjugates compared to nativechymotrypsin, but GM was able to inhibit chymotrypsin-pQA conjugatesmore quickly than native chymotrypsin (FIG. 18). As shown in Table 10below, AP Ki values were over 27 fold higher for chymotrypsin-pQA₁₉₈compared to native chymotrypsin, and GM Ki values were almost 4 foldhigher for native chymotrypsin than chymotrypsin-pQA₁₉₈.

TABLE 10 Inhibition constants of aprotinin and glycine max for nativeand chymotrypsin-pQA198 at 25° C. Inhibitor Sample Ki (nM) AprotininNative CT 56.3 CT-pQA₁₉₈ 1560 Glycine Max Native CT 172.0 CT-pQA₁₉₈ 46.1

It was hypothesized that electrostatic attraction and repulsion wereresponsible for the change in inhibition kinetics seen forchymotrypsin-pQA conjugates. As AP was positively charged, electrostaticrepulsion between the high density cationic pQA shell surrounding CT andAP pushed the inhibitor molecule away from the enzyme active site.Conversely, electrostatic attraction between pQA and the negativelycharged GM pulled the inhibitor toward the active site. These repulsiveand attractive forces would be expected to influence the localconcentration of inhibitor in the region of the active site.

In addition to having opposite surface charges, AP is a completiveinhibitor, whereas GM is a noncompetitive inhibitor. Competitiveinhibitors, which function by competing for the enzyme active site withthe natural substrate, will increase K_(M) values while not affectingk_(cat) during inhibition. GM, a mixed noncompetitive inhibitor, caninhibit by binding to the active site and other locations on CT'ssurface, causing both K_(M) and k_(cat) to change during inhibition.Chymotrypsin-pQA conjugates showed the same changes in k_(cat) and KM aswould be expected with each type of inhibition (FIGS. 23/25). Thus,while the cationic pQA shell did predictably manipulate inhibitionkinetics of CT, it is likely that the mechanism of binding is still thesame as native chymotrypsin.

Conclusions

In this series of experiments, protein-initiated ATRP was used tosynthesize chymotrypsin-pQA conjugates of four molecular weights with adense cationic shell surrounding each enzyme molecule. In support of thehypothesis behind this series, these conjugates changed enzyme activity,substrate affinity, stability, and inhibitor binding in the mannerpredicted by the hypothesis. Enzyme activity for chymotrypsin-pQAconjugates at low pH was increased compared to native CT due to chargestabilization of the active site catalytic triad. Substrate affinity atpH 5-10 was increased for chymotrypsin-pQA conjugates due to favorablepartitioning effects between the cationic shell and the hydrophobicsubstrate. Enzyme stability was increased at elevated temperature andlow pH after pQA conjugation due to both structural stabilization andsteric hindrance of autolysis by pQA. The inhibition kinetics of twochymotrypsin inhibitors, aprotinin (competitive) and chymotrypsininhibitor from glycine max (mixed non-competitive), were also modifiedby electrostatic attraction and repulsion between the cationic shell andthe inhibitor. It has been shown in this study that many differentenzyme properties as well as protein-protein interactions can be tunedfor a desired purpose using polymer-based protein engineering.

In further support of the foregoing series, the NMR peaks in FIGS. 19and 20 showed that no degradation of the polymer occurred during theacid hydrolysis procedure. NMR spectra peaks in FIG. 19 are slightlybroader due to the overlapping of chymotrypsin peaks. Large peaks thatwould be assigned to chymotrypsin cannot be seen in FIG. 19, because itis hidden due to its orientation in the center of the dense polymershell. ¹H NMR spectra of chymotrypsin-pQA conjugates and cleaved pQAfrom the conjugates, GPC traces of the cleaved pQA, Table 8 showing theMichaelis-Menten kinetic values of hydrolysis of Suc-AAPF-pNA atdifferent pH, and first (V_(i) versus [S]₀ on hydrolysis ofSuc-AAPF-pNA) and secondary plots (K_(M app)/V_(max app) versus [I]₀)for determination of dissociation constant of enzyme-inhibitorcomplexes. FIGS. 21, 22 and 24 plot additional data and Tables 11, 12,13 and 14 and apparent K_(M) and V_(max) values for native chymotrypsinand hymotrypsin-pQA₂₀₀ incubated with aprotinin or GM at 25° C. in 0.1 Msodium phosphate buffer (pH 8.0).

TABLE 11 Apparent K_(M) and V_(max) values for native chymotrypsinincubated with aprotinin at 25° C. in 0.1M sodium phosphate buffer (pH8.0) [I]0 (mM) 0 0.098 0.196 0.294 0.392 0.490 K_(M) (mM) 85.9 174.9276.6 374.7 450.3 645.8 V_(max) (mM/sec) 1.13 1.17 1.14 1.19 1.04 1.13K_(M)/V_(max) (sec−1) 76.3 149.2 242.8 314.3 431.6 569.45

TABLE 12 Apparent KM and V_(max) values for chymotrypsin-pQA₂₀₀incubated with aprotinin at 25° C. in 0.1M sodium phosphate buffer (pH8.0) [I]0 (mM) 0 0.98 1.96 2.94 3.92 KM (mM) 46.4 69.6 106.4 113.6 160.6V_(max) (mM/sec) 0.79 0.77 0.82 0.74 0.78 KM/V_(max) (sec−1) 58.9 90.1129.4 152.9 204.6

TABLE 13 Apparent K_(M) and V_(max) values for native chymotrypsinincubated with GM at 25° C. in 0.1M sodium phosphate buffer (pH 8.0)[I]0 (mM) 0 0.049 0.098 0.147 0.196 0.294 K_(M) (mM) 78.7 94.9 120.5182.1 196.5 213.4 V_(max) (mM/sec) 1.10 1.05 1.18 1.21 1.23 1.14K_(M)/V_(max) (sec−1) 71.8 90.4 102.2 150.1 159.9 188.0

TABLE 14 Apparent K_(M) and V_(max) values for chymotrypsin-pQA₂₀₀incubated with GM at 25° C. in 0.1M sodium phosphate buffer (pH 8.0)[1]0 (mM) 0 0.049 0.098 0.147 0.196 0.294 K_(M) (mM) 46.4 94.0 120.5141.0 165.7 242.4 V_(max) (mM/sec) 0.79 0.74 0.68 0.60 0.61 0.56K_(M)/V_(max) (sec−1) 58.9 125.9 177.4 236.5 273.6 435.3Effect of pQA Conjugation on Aprotinin Binding to Chymotrypsin

The electrostatic repulsion described in the discussion section requiredthe inhibitor concentration to be approximately 10 fold higher (0-3.92μM) for chymotrypsin-pQA conjugates compared to native chymotrypsin whendetermining apparent and KM values.

Experimental Series IV Relating to Use of Dual Block Polymer-BasedProtein Engineering to Increase pH and Temperature Stability

In Experimental Series IV, dual temperature responsiveCT-pSBAm-block-pNIPAm conjugates with different polymer chain lengthsand molecular weights were synthesized using a “grafting from” approachwith two successive ATRP reactions. The NHS-functionalized Amide ATRPinitiator halide was chlorine.

Synthesis of N-2-chloropropionyl-β-alanine N′-oxysuccinimide ester (1)

A mixture of 2-chloropropionyl chloride (9.7 mL, 100 mmol) and1,4-dioxane (50 ml) was slowly added into a solution of β-alanine (8.9g, 100 mmol) and sodium hydrogen carbonate (21 g, 250 mmol) in mixtureof deionized water (200 mL) and 1,4-dioxane at 0° C. The mixture wasstirred at room temperature for 2 h. The water phase was washed withdiethyl ether (100 mL×3) and adjusted to pH 2 with 1.0 N HCl aq. at 0°C. The product was extracted with ethyl acetate (150 mL×6). The organicphase was dried with MgSO₄ and evaporated to remove solvent.N-2-chlorolpropionyl-β-alanine was isolated by recrystallization from amixture of diethyl ether and n-hexane (1:1 volume ratio); yield 6.8 g(38%), mp 102-105° C. ¹H NMR (300 MHz, CDCl₃) δ 1.73 (d, 3H, J=6.9 Hz,NHC═OCHCH₃Cl), 2.66 (t, 2H, J=6.3 Hz, HOOCCH₂CH₂NHC═O), 3.58 (q, 2H,J=6.3 Hz, HOOCCH₂CH₂NHC═O), 4.44 (q, 1H, J=6.9 Hz, NHC═OCHCH₃Cl) and7.19 (broad s, 1H, amide proton) ppm. IR (KBr) 3290, 3097, 2980, 2919,1701, 1657, 1567, 1443, 1370, 1299, 1223, 1078, 988, 936 and 670 cm⁻¹.

N,N′-diisopropylcarbodiimide (3.0 g, 24 mmol) was slowly added to thesolution of N-2-chloropropionyl-β-alanine (3.6 g, 20 mmol) andN-hydroxysuccinimide (2.8 g, 24 mmol) in dichloromethane (100 mL) at 0°C. The mixture was stirred at room temperature overnight. Afterfiltration, the solution was evaporated to remove solvent.N-2-chloropropionyl-β-alanine. N′-oxysuccinimide ester (III) waspurified by recrystallization from 2-propanol with a yield of 4.5 g(82%), mp 93-97° C. ¹H NMR (300 MHz, CDCl₃) β 1.73 (d, 3H, J=6.9 Hz,NHC═OCHCH₃Cl), 2.87 and 2.89 (s and t, 4H and 2H, J=6.6 Hz, ethylene ofsuccinimide and NHSOOCCH₂CH₂NHC═O), 3.70 (t, 2H, J=6.6 Hz,NHSOOCCH₂CH₂NHC═O), 4.41 (q, 1H, J=6.9 Hz, NHC═OCHCH₃Cl), and 7.13,(broad s, 1H, amide proton). IR (KBr) 3369, 2991, 2934, 1813, 1782,1729, 1671, 1560, 1449, 1420, 1381, 1297, 1218, 1073, 992, 885, 810, 655and 597 cm⁻¹.

Calculation of ATRP Initiator Immobilization on Enzyme Molecule

FIG. 27 shows the MALDI-TOF-MS spectra for native chymotrypsin and ATRPinitiator modified chymotrypsin. Calculated M_(n) values were 25493 Dafor native CT and 28534 Da for CT-initiator-Cl. From these values,calculations indicated that there were 15 initiating sites for everychymotrypsin molecule. The presence of fifteen initiating sites perchymotrypsin molecule indicates that all of the 14 lysines on CT as wellas the N-terminus were modified using this technique. From the MALDIspectra, two peaks were seen for native CT (one large intensity peak andone lower intensity) and this shape is conserved in the CT-initiator-Clmolecule as well. However, the peak for CT-initiator-Cl is broader thannative CT, indicating the macroinitiator molecules are not completelymonodisperse. Thus, it was estimated that each chymotrypsin molecule hadbetween 13-15 initiating molecules, or an average of 14 polymers perenzyme molecule.

Reaction between the ATRP Initiator and Chymotrypsin

Synthesis of the ATRP initiating molecules was carried out as shown inScheme IV.

Following synthesis, initiator molecule (194 mg, 0.7 mmol) and CT (500mg, 0.02 mmol protein, 0.32 mmol primary amine) were dissolved in 0.1 Msodium phosphate buffer (pH 8.0). The solution was stirred at 4° C. for4 hours, and then dialyzed against deionized water, using dialysistubing with a molecular weight cut off of 15 kDa, for 24 hours at 4° C.and then lyophilized.

Surface Initiated ATRP from CT-Initiator

To synthesize CT-pSBAm-block-pNIPAm conjugates, first the CT-NHS-Clfunctionalized initiator conjugate (50 mg, 0.029 mmol initiator) andSBAm [335 mg (1.2 mmol), 525 mg (1.8 mmol), 701 mg (2.4 mmol)] weredissolved in 0.1 M sodium phosphate buffer (20 mL, pH 6.0) with 35 mgNaCl (30 mM). In a separate flask, Me6TREN (33 μL, 0.12 mmol) wasdissolved in deionized water (5 mL) and bubbled with argon for 10 min.Cu(I)Cl (17 mg, 0.12 mmol) was added to the Me6TREN solution and Argonwas bubbled for an additional 50 minutes prior to addition of the coppercatalyst solution to the monomer solution. After combining the twosolutions, the reaction mixture was stirred for 5 h at 25° C. until thereaction was stopped by exposing the solution to air. Lastly, thesolution was purified using dialysis tubing with a molecular weightcutoff (MwCO) of 25 kDa for 48 hours against deionized water at 4° C.and then lyophilized.

Following initial synthesis of CT-pSBAm conjugates of different chainlengths, pNIPAm was grown from CT-pSBAm using chain extension to yieldCT-pSBAm-block-pNIPAm conjugates. CT-pSBAm conjugates [200 mg of CT-35(0.02 mmol initiator), 280 mg of CT-50 (0.02 mmol initiator), 350 mg ofCT-90 (0.01 mmol initiator)] and NIPAm [108 mg (0.96 mmol), 163 mg (1.44mmol), 135 mg (1.2 mmol)] were dissolved in 0.1 M sodium phosphatebuffer (20 mL, pH 6.0) with 35 mg NaCl (30 mM) and bubbled with argon.In a separate flask Me6TREN [10.7 μL (0.05 mmol), 10.7 μL (0.05 mmol),6.4 μL (0.03 mmol)] was dissolved in deionized water (5 mL) and bubbledwith Argon for 10 min. Cu(I)Cl (4 mg (0.04 mmol), 4 mg (0.04 mmol), 2.4mg (0.03 mmol)) was added to the Me6TREN solution and argon was bubbledfor an additional 50 minutes. The Me6TREN/CuCl solution was quicklytransferred to the CT-pSBAm/NIPAm solution and reaction was allowed toproceed for 5 h at 25° C. The reaction was stopped by quenching with airand the reaction mixture was purified using dialysis tubing with MwCO 25kDa for 48 h against deionized water at 4° C., and then lyophilized.

UCST Cloud Point Curves for CT-pSBAm Conjugates

CT-pSBAm conjugates (CT-50-closed diamond, CT-75-closed circle,CT-100-closed square) were dissolved in 0.1 M phosphate buffer (pH 8.0)at 3 mg/mL. Samples were placed in quartz cuvette and were cooled from35° C. or 25° C. to 5° C. at 0.5° C./min. Absorbance at 490 nm (solutionturbidity) for each CT-pSBAm conjugate was measured in increments of 1°C. UCST phase transition for CT-pSBAm conjugates was dependent onpolymer molecular weight. Phase transitions were quicker and absoluteabsorbance measurements after phase transition were higher for CT-pSBAmconjugates compared to CT-pSBAM-b/ock-pNIPAm conjugates. (FIG. 27)

Method to Determine M_(n) of Second Block pNIPAm

Due to the contrasting size of pNIPAm and pSBAm (pSBAm monomer unit ismuch larger than that of pNIPAm), there was not an appropriate standardfor GPC analysis. Thus, M_(w) and M_(n) for pSBAm-block-pNIPAm polymerscould not be calculated using GPC techniques. Instead of GPC, M_(n)values for chain extended second block pNIPAm was determined usingrelative ratios of peaks in NMR spectra. M_(n) for each of the secondblock pNIPAm segments were estimated using the NMR spectra of diblockpolymers cleaved from chymotrypsin (FIGS. 29, 30, 31). To do this, therelative intensity from peak c (3.8-4.0 ppm), corresponding to oneproton in the pNIPAm block was compared with the broad signal complexfrom 0.8-2.3 ppm (signals a+b+d+e+f+g+h) corresponding to 18 totalprotons in both the pNIPAm and pSBAm blocks. From these ratios, theM_(n) of the pNIPAm block was calculated when comparing to the for pSBAmcalculated from GPC analysis.

Polymer Cleavage from CT Surface

Both pSBAm and pSBAm-block-pNIPAm were cleaved from the surface ofCT-polymer conjugates using acid hydrolysis. CT-pSBAm conjugates wereincubated (15 mg/mL) in 6N HCl at 110° C. under vacuum for 24 hours.CT-pSBAm-block-pNIPAm (20 mg/mL) conjugates were incubated in 4.5Np-toluene sulfonic acid at 80° C. under vacuum for 72 hours. Followingincubation, cleaved polymers were isolated from CT using dialysis tubing(MwCO 1K Da) for 48 hours and then lyophilized.

Characterization of Cleaved Polymers

Number and weight average molecular weights (M_(n) and M_(w)) and thepolydispersity index (M_(w)/M_(n)) were estimated by gel permeationchromatography (GPC) for pSBAm polymers cleaved from CT. Analysis wasconducted on a Water 2695 Series with a data processor, using 80/20mixture of 0.1 M sodium phosphate buffer (pH 9.0) and acetonitrile with0.01 volume % NaN₃ as an eluent at a flow rate 1 mL/min, with detectionby a refractive index (RI) detector. Polystyrene sulfonate standardswere used for calibration. M_(n) was calculated for pSBAm-block-pNIPAmcleaved from CT by quantitatively comparing NMR peaks (integration ofpeaks) of copolymer to cleaved first block pSBAm NMR spectra.

LCST/UCST Determination

CT-pSBAm-block-pNIPAm conjugates (2-3 mg polymer/mL) were dissolved in0.1 M phosphate buffer (pH 8.0) in quartz cuvette. Conjugates werecooled from 25° C. to 1° C. and then heated up to 40° C. at ±0.5°C./min. The absorbance at 490 nm was measured in 1° C. increments andLCST/UCST temperature was calculated from the inflection point in thecloud point curves.

CT and CT Conjugate Biocatalytic Activity

N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide was used as asubstrate for enzyme bioactivity assays. In a cuvette, 0.1 M sodiumphosphate buffer (2820-2970 μL, pH 8.0), substrate (0-150 μL, 6 mg/mL inDMSO (0-500 μM)), and enzyme (30 μL, 0.1 mg enzyme/mL 0.1 M pH 8.0sodium phosphate buffer (0.04 μM)) were mixed. The rate of thehydrolysis was determined by recording the increase in absorbance at 412nm for the first 30 seconds after mixing. K_(M) and k_(cat) values werecalculated using EnzFitter software when plotting substrateconcentration versus initial hydrolysis velocity.

Thermal Stability

Native CT and CT-conjugates (40 μM) were dissolved in 0.1 M sodiumphosphate buffer (pH 8.0) and incubated in a water bath in 50 μLaliquots at 37° C. At specified time points, aliquots were removed anddiluted to 4 μM using 0.1 M sodium phosphate buffer (pH 8.0). Residualactivity, measured in 0.1 M sodium phosphate buffer (pH 8.0) at 25° C.,was calculated as the ratio of activity remaining relative to theactivity at time zero. Substrate (Suc-AAPF-pNA) concentration was keptconstant at 288 μM for each sample and time point. Native CT andconjugate activities were measured in duplicate at each time point.

In Vitro Gastric Acid Stability

Native CT and CT-conjugates were incubated at 4 μM in 167 mM HCl at 37°C. in 50 μL aliquots. Aliquots were removed at specified time points andresidual activity was measured at 25° C. in 0.1 M sodium phosphatebuffer (pH 8.0) with Suc-AAPF-pNA as substrate (288 μM). Each time pointwas measured in duplicate and residual activity was calculated as theratio of activity remaining from time zero.

Stability to Pepsin Degradation

Native CT and CT-conjugates (4 μM) were incubated in 167 mM HCl with 16nM pepsin at 37° C. in 50 μL aliquots. Samples were retrieved atspecified time points and residual activity was measured in 0.1 M sodiumphosphate buffer (pH 8.0) at 25° C. with Suc-AAPF-pNA as substrate (288μM). Each time point was measured in duplicate and residual activity wascalculated as the ratio of activity remaining from time zero. As acontrol, pepsin (16 nM) bioactivity towards Suc-AAPF-pNA was measured atpH 8.0 and no product formation was observed.

Size Measurements During 167 mM HCl (pH 1) Incubation

CT conjugates (4 μM) and native CT (29 μM) passed through a 0.2 μMcellulose filter were incubated in 167 mM HCl at 37° C. in 1 mLaliquots. Aliquots were removed from incubation at each specified timepoint. Hydrodynamic diameter was then determined using a Malvernzetasizer nano-ZS at 25° C. Intensity PSD measurements, averaged overfive sample runs at each time point, were used to calculate hydrodynamicdiameter (D_(h)).

Results and Discussion

Conjugate Synthesis and Polymer Characterization

As described above, separate CT-pDMAPS and CT-pNIPAm conjugates withtemperature responsiveness were synthesized by “grafting from” a watersoluble bromine functionalized ATRP initiator coupled to chymotrypsin(CT-Br). In this study, a similar water soluble initiating molecule(Ini-Cl), functionalized with chlorine rather than bromine, wasconjugated to chymotrypsin to yield the chymotrypsin ATRP macroinitiator(CT-Cl) (Scheme IV). Similar to Ini-Br, the Ini-Cl molecule wasfunctionalized with an NHS-ester to react with primary amines on surfacelysines and the N-terminus of CT. Following synthesis of the CT-Clmacroinitiator, pSBAm was first grown from the surface of CT with threedifferent molecular weights, yielding three conjugates with UCSTbehavior (CT-35, CT-50, CT-90). From GPC chromatograms (not shown), itwas determined no residual free chymotrypsin was left after first blocksynthesis. The synthesis of CT-pNIPAm-block-pSBAm conjugates wasexplored, but sequential ATRP reactions in this order were not possiblewithout optimization. After purification of CT-pSBAm conjugates, chainextension with pNIPAm was completed to yield three CT-pSBAm-block-pNIPAmconjugates with three different molecular weights that showed both UCSTand LCST behavior (CT-35/39, CT-50/67, CT-90/100). The nomenclature usedfor each of the conjugates corresponds to degree of polymerization ofeach conjugate based on GPC and NMR analysis of cleaved polymers (notshown). The use of Ini-Cl/Cu(I)Cl, the addition of NaCl to thepolymerization solution, and short polymerization time helped to lowerthe PDI of polymers grown from the surface of CT in this study (Table15).

TABLE 15 Molecular weight and hydrodynamic diameter ofCT-pSBAm-block-pNIPAm conjugates Cleaved CT Polymer Conjugate PDI MW(M_(w)/ GPC/ Size (D_(h)) Sample M_(n) M_(n)) BCA NMR [nm] CT-pSBAm₃₅10.2 kDa 1.59 173 kDa 171 kDa 24.2 ± 2.5 CT-pSBAm₅₀ 15.7 kDa 1.43 248kDa 248 kDa 22.7 ± 1.9 CT-pSBAm₉₀ 26.2 kDa 1.86 362 kDa 395 kDa 23.3 ±1.0 CT-pSBAm₃₅- 14.6 kDa — 302 kDa 232 kDa 46.1 ± 4.5 block-pNIPAm₃₉CT-pSBAm₅₀- 23.3 kDa — 427 kDa 354 kDa 47.6 ± 8.7 block-pNIPAm₆₇CT-pSBAm₉₀- 37.5 kDa — 475 kDa 553 kDa 64.1 ± 4.5 block-pNIPAm₁₀₀

It is also likely the reaction conditions reduced growing chaintermination and ligand/catalyst degradation, which conserved the livingnature of the polymer chain to allow chain extension of pNIPAm fromCT-pSBAm (Simakova, A.; Averick, S. E.; Konkolewicz, D.; Matyjaszewski,K. Macromolecules 2012, 45, 6371; Tsarevsky, N. V.; Pintauer, T.;Matyjaszewski, K. Macromolecules 2004, 37, 9768.). Gas chromatogramresults (not shown) indicated that minimal amounts (<10%) of CT-pSBAmconjugates did remain after chain extension indicating some chaintermination might have occurred during first block synthesis.

Phase Transition Temperatures of CT-pSPAm-block-pNIPAm Conjugates

UCST and LCST temperatures for each of the three molecular weightconjugates (CT-35/39, CT-50/67, and CT-90/100) were determined bymeasuring solution turbidity (absorbance at 490 nm) at temperatures from0-40° C. Each of the conjugates displayed both LCST and UCST behavior,but the specific phase change temperature was dependent upon the polymerchain length (FIG. 10). Each of the conjugates showed LCST behavior at29° C. in 0.1 M phosphate buffer (pH=8.0). As previously reported,linear pNIPAm LCST is independent of molecular weight when the molecularweight is not ultra-high²³, so this result was not unexpected. LCSTphase transition at 29° C. was slightly lower than previously reportedLCST values for pNIPAm, but the lowered value is consistent with LCSTbehavior in salt buffers compared to deionized water (Zhang, Y.; Furyk,S.; Sagle, L. B.; Cho, Y.; Bergbreiter, D. E.; Cremer, P. S. J. Phys.Chem. C 2007, 111, 8916). The LCST also agreed well with the LCSTtemperature determined in Experimental Section II for CT-pNIPAmconjugates. The turbidity of these solutions (0.2-0.25 AU) was alsolower than CT-pNIPAM conjugates. When above the LCST, pNIPAm polymerchains are insoluble in aqueous solutions and thermodynamically preferto minimize interactions with water. Thus, as each of the conjugatesreached temperatures above 29° C., the pNIPAm component of thebioconjugate collapsed. For free pNIPAm in solution, large aggregatesform; greatly increasing turbidity. However, for CT-pSBAm-block-pNIPAm,hydrophilic CT and pSBAm components, even at temperatures above theLCST, prevented more extreme aggregation, which caused turbiditymeasurements above the LCST to plateau at a lower value than free pNIPAMor CT-pNIPAm conjugates.

When comparing CT-pSBAm-block-pNIPAm cloud point curves with CT-pSBAmcurves (FIG. 28), it was clear that the pNIPAm block influenced the UCSTbehavior of the pSBAm component in the final bioconjugate. A lowertemperature was required for the CT-pSBAm-b/ock-pNIPAm conjugates toshow collapsed, insoluble behavior compared with CT-pSBAm conjugates. Atlow temperature, pSBAm was hydrophobic, but the pNIPAm and CT componentsof the conjugate were still hydrophilic and influenced the overallbehavior of the conjugate. In addition to polymer collapse, aggregationof hydrophobic pSBAm polymer blocks between different CT moleculescontributed to the turbidity seen in the cloud point curves. The pNIPAmblock, located on the outside of the CT conjugates, likely stericallyhindered pSBAm association between CT molecules, which could lower thetransition temperature onset as determined by cloud point curves. TheUCST behavior for CT-90/100, the longest chain conjugate, did not show asharp increase in absorbance at a specific temperature. Instead,turbidity measurements for CT-90/100 increased linearly when decreasingtemperature from 15-3° C., and ultimately showed a sharp increase inabsorbance around 2° C. As with CT-35/39 and CT-50/67, it washypothesized that this effect was due to steric hindrance of pNIPAm andthe hydrophilicity of CT and pNIPAm at this temperature range. However,the long chain length of pNIPAm on the outside of CT-90/100 prevented asharp increase in turbidity at the temperature where insolubility wasinitially observed.

Effect of Double Shelled Polymer-Based Protein Engineering on CTBioactivity

CT conjugate kinetics were examined at a variety of temperatures (2.5°C., 7° C., 16.5° C., 24.5° C., 33° C., and 37.5° C.) to determine theeffect of polymer UCST and LCST phase transitions and molecular weighton enzyme kinetics (see FIGS. 11(a)-(c) and Table 16 below).

TABLE 16 Michelis-Menten kinetics for CT and CT conjugate catalyzedhydrolysis of NS-AAPF-pNA from zero to 37.5° C. K_(M) k_(cat)k_(cat)/K_(M) (K_(M)) × (k_(cat)) × (k_(cat)/K_(M)) × Sample [μM][sec⁻¹] [sec⁻¹/μM] (K_(M))_(CT) (k_(cat))_(CT) (k_(cat)/K_(M))_(CT) 2.5°C. Native CT 30 ± 5.1 8.9 ± 0.4 0.33 ± 0.06 — — — CT-35/39 47 ± 7.5 6.3± 0.5 0.14 +0.02 1.59 0.71 0.42 CT-50/67 49 ± 7.7 6.8 ± 0.2 0.14 ± 0.021.65 0.76 0.42 CT-90/100 49 ± 10 4.5 ± 0.4 0.08 ± 0.02 1.66 0.51 0.24 7°C. Native CT 29 ± 6.9 9.7 ± 0.5 0.34 ± 0.09 — — — CT-35/39 41 ± 9.6 7.8± 0.5 0.19 ± 0.05 1.42 0.80 0.57 CT-50/67 41 ± 7.3 8.7 ± 0.5 0.21 ± 0.061.45 0.90 0.62 CT-90/100 45 ± 9.6 6.5 ± 0.4 0.15 ± 0.03 1.58 0.67 0.4316.5° C. Native CT 37 ± 8.6 16 ± 0.9 0.43 ± 0.10 — — — CT-35/39 41 ± 6.813 ± 0.5 0.31 ± 0.05 1.09 0.79 0.72 CT-50/67 39 ± 5.6 14 ± 0.5 0.36 ±0.05 1.04 0.87 0.84 CT-90/100 43 ± 7.1 10 ± 0.5 0.24 ± 0.04 1.16 0.640.56 24.5° C. Native CT 51 ± 8.7 25 ± 1.3 0.50 ± 0.10 — — — CT-35/39 50± 10 22 ± 0.9 0.43 ± 0.08 0.98 0.86 0.87 CT-50/67 53 ± 7.3 21 ± 0.9 0.41± 0.06 1.03 0.82 0.92 CT-90/100 50 ± 7.8 16 ± 0.7 0.31 ± 0.05 0.97 0.620.63 33° C. Native CT 57 ± 5.0 36 ± 1.3 0.64 ± 0.08 — — — CT-35/39 67 ±5.3 32 ± 0.9 0.47 ± 0.04 1.18 0.88 0.75 CT-50/67 65 ± 7.2 33 ± 1.4 0.51± 0.07 1.14 0.90 0.80 CT-90/100 75 ± 6.9 25 ± 0.7 0.33 ± 0.03 1.32 0.680.51 37.5° C. Native CT 59 ± 6.9 47 ± 1.7 0.80 ± 0.10 — — — CT-35/39 71± 6.7 43 ± 1.4 0.61 ± 0.06 1.19 0.92 0.77 CT-50/67 71 ± 6.3 43 ± 1.30.61 ± 0.06 1.21 0.92 0.76 CT-90/100 75 ± 11 32 ± 1.6 0.43 ± 0.07 1.270.69 0.54

The rate of hydrolysis ofN-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide by CT and CTconjugates in 0.1 M sodium phosphate buffer (pH 8.0) was used todetermine the impact of doubled-shelled PBPE on CT bioactivity. CTconjugate bioactivity was temperature dependent after conjugation ofpSBAm-block-pNIPAm. Relative K_(M) and k_(cat) values for CT-35/39,CT-50/67, and CT-90/100 were all modified after conjugation, but onlyrelative K_(M) values were dependent upon temperature. For CT-35/39 andCT-50/67, relative k_(cat) values were between 0.8 and 0.9 at each ofthe tested temperatures. Relative k_(cat) values for CT-90/100 wereslightly lower than CT-35/39 and CT-50/67 conjugates, but similarly,enzyme activity was conserved and was independent of temperature. Inexperiments described in Experimental Section III herein relating to themethods of the invention described herein, long chain polymerconjugation has decreased k_(cat) values due to limited structuralflexibility, which likely contributed to decreased k_(cat) values inthis study as well (Rodríguez-Martínez, J. A.; Solá, R. J.; Castillo,B.; Cintrón-Colón, H. R.; Rivera-Rivera, I.; Barletta, G.; Griebenow, K.Biotechnol. Bioeng. 2008, 101, 1142).

While k_(cat) values were independent of temperature, relative K_(M)values for CT-pSBAm-block-pNIPAm bioconjugates showed a significantdependence to both low and high temperatures. At 25° C., each of theconjugates calculated K_(M) values were similar to native CT. However,at temperatures both higher and lower than 25° C., relative K_(M) valuesincreased significantly for each of the conjugates. At low temperaturesthe increase in relative K_(M) was more extreme than the K_(M) increaseat high temperature. At assay temperatures below 25° C., relative K_(M)values increased until reaching a maximum (˜1.6-1.7 for each of theconjugates) at 2.5° C. At 40° C., relative K_(M) for each of theconjugates was approximately 1.2, indicating lower substrate infinityfor the conjugates compared to native CT at this temperature. ForCT-35/39 and CT-50/67, relative K_(M) values were slightly lower at 35°C. than K_(M) values at 40° C. CT-90/100 relative K_(M) values were onlyslightly higher at 35° C. than 40° C.

It was hypothesized that the increase in K_(M) values at both high andlow temperature was a result of restricted access to the active site forthe model substrate due to steric hindrance caused by polymer collapseduring both UCST and LCST phase transitions. FIG. 12 shows thehypothesis of how polymer collapse impacted substrate access to theenzyme active site, drawn to the scale of each polymer blockapproximated from dynamic light scattering data (Table 15 of thisSection). At temperatures above 29° C., as determined by cloud pointcurves, the pNIPAm block of the polymer was collapsed upon itself tominimize its interaction with water. As the pNIPAm polymers collapsed, amore compact shell existed compared to pNIPAm orientation at 25° C.,which likely restricted the model substrate's ability to reach theactive site. At low temperature, it was hypothesized that the increasein relative K_(M) was also due to steric hindrance from polymercollapse, except the pSBAm polymer block collapsed at low temperaturerather than pNIPAm. As seen from the cloud point curves, CT-90/100 UCSTpolymer collapse began at 16° C., so it was not surprising to see thehighest relative K_(M) increase of the three conjugates at this assaytemperature. As temperature decreased, the UCST induced polymer collapseappeared to continue to increase as evidenced by the increase inturbidity measurements in cloud points curves. The increase seen inrelative K_(M) values at lower temperatures was most likely due to thisincrease in polymer collapse and dehydration. Once the substrate reachedthe active site, the rate of the reaction was similar at eachtemperature measured, as shown by the lack of dependence of relativek_(cat) values on temperature.

Chain length of the polymers did not appear to have a large effect onthe relative K_(M) values. While CT-90/100 conjugates did have slightlyhigher relative K_(M) values than CT-35/39 and CT-50/67, a clear trendbetween the three conjugates was not noticeable. It is also important tonotice the difference in relative K_(M) values with respect to thelocation of the collapsed polymer in the conjugate. Since the pSBAmblock was synthesized first, this polymer was closer to the core of theconjugate, while the pNIPAm block was on the outside of the conjugates.It was surmised that, due to this orientation, polymer collapse andturbidity increases were seen at both high and low temperatures, but thespecific geometry of the overall collapsed conjugate was different. Itwas hypothesized that the pSBAm collapse showed higher relative K_(M)values due to its location closer to the core of the conjugate (closestto the active site). In addition, while pNIPAm collapse at hightemperature also induced increased relative K_(M) values, the effect wasnot as pronounced due to its location on the outside of the conjugate.

Relative productivity (k_(cat)/K_(M)) ratios for each of theCT-pSBAm-block-pNIPAm conjugates were also dependent upon temperature.Relative productivity values for CT-35/39 and CT-50/67 were similar tonative CT at 25° C., and CT-90/100 was slightly reduced due to lowerk_(cat) values at this temperature. For each temperature tested otherthan 25° C., k_(cat)/K_(M) values were decreased, mostly as a result ofthe increased relative K_(M) values seen after phase transitions. Thelargest decrease in productivity ratios was seen at 2.5° C., whererelative K_(M) values were the highest and a slight decrease in k_(cat)values was seen for each conjugate.

CT Conjugate Stability

The thermal stability, pH stability, and protease degradation stabilityconditions of the CT-pSBAm-block-pNIPAm conjugates were explored indetail. CT conjugates of each molecular weight had higher stability thannative CT to (a) incubation at 37° C., (b) incubation in 167 mM HCl (pH1), and (c) incubation with pepsin (FIG. 13). Specifically, maintainingstability of proteins as they are subjected to extreme pH and proteasedegradation (as would be seen in the GI tract) is a large challenge.Most studies on oral peptide delivery technologies have focused ontransport through the intestinal membrane (Reineke, J.; Cho, D. Y.;Dingle, Y. L.; Cheifetz, P.; Laulicht, B.; Lavin, D.; Furtado, S.;Mathiowitz, E. J. Controlled Release 2013, 170, 477) orpharmacokinetics/biodistribution, (Xu, Q.; Boylan, N. J.; Cai, S.; Miao,B.; Patel, H.; Hanes, J. J. Controlled Release 2013, 170, 279.) but donot examine stability of the protein in the GI tract.

Stability at Ambient Temperature and Neutral pH

CT conjugates lost only 10% of their activity after 8 hours incubationat 40 μM in 0.1 M sodium phosphate buffer (pH 8.0), while native CT losthalf of its activity over the same time period (FIG. 13a ). At 37° C.and pH 8, CT was still active and, consequently, one contributor toirreversible inactivation at this temperature and pH was autolysis. As aprotease, CT hydrolyzes peptide bonds to break down proteins, and CTinactivates itself due to self-digestion of unfolded CT in solution. Asa result of the polymer density around CT conjugates, steric hindrancelimited CT molecules access to each other, decreasing autolysis andincreasing stability. Previously, PEGylation of protein molecules hasbeen shown to reduce structural dynamics (Rodríguez-Martinez, J. A.;Solá, R. J.; Castillo, 13.; Cintrón-Colón, H. R.; Rivera-Rivera, I.;Barletta, G.; Griebenow, K. Biotechnol. Bioeng. 2008, 101, 1142), andcharged polymers have increased the stability of CT after conjugationdue to charge effects (Keefe, A. J.; Jiang, S. Y. Nat. Chem. 2012, 4,60; Baldwin, R. L. Biophys. J. 1996, 71, 2056). Increased stabilityimparted by CT-pSBAm-block-pNIPAM conjugates was likely due to acombination of both effects as the pSBAm block contained proteinstabilizing ions and pNIPAm chemical structure was similar to PEG.

Stability at Extremes of pH

Interestingly, CT conjugates also showed increased to stability to lowpH. Native CT and CT conjugates were incubated in 167 mM HCl (pH 1) at37° C. for 3 hours to mimic gastric acid. In vivo, gastric acid promotesunfolding of proteins to increase access of pepsin to cleavable aminoacid sequences, Each of the CT conjugates maintained at least 60% ofactivity after incubation in 167 mM HCl for 3 hours, compared tocomplete activity loss for native CT after the same time period (FIG.13b ). In 167 mM HCl, (pH 1), native CT unfolds due to disruption ofhydrogen bonding. One can imagine two mechanisms through which PBPEmight stabilize CT to such a dramatic extent. First, the polymerstabilized the structure of CT, which reduced unfolding. Second, as someCT molecules unfolded, access to cleavage sites by autolysis wasrestricted by steric hindrance of polymer as seen at 37° C. and pH 8.0.However, at pH 1 the enzyme should be inactive, and autolysis cannotcontribute to CT inactivation. The activity of CT and CT-conjugates wasmeasured at pH 1.0 and no product formation was observed with largeexcess of substrate (data not shown). Thus, protein unfolding withoutautolysis was the main mechanism for CT inactivation in 167 mM HCl. Tofurther develop the mechanistic understanding, dynamic light scatteringwas used to examine native CT and CT conjugate hydrodynamic diameterduring incubation at pH 1 (FIG. 14). After 2 hours, the hydrodynamicdiameter (D_(h)) for native CT increased 9-fold, while CT conjugate sizeincreased less than 20%. After incubation at low pH in 167 mM HCl,hydrogen bonding and other forces that maintain globular structure ofproteins were likely disrupted for native CT. The disruption of forcesled to protein unfolding, causing the protein to transition from aglobular conformation to a more linear structure, which increased D_(h)measurements for native CT. While very small increases in D_(h) wereseen for CT conjugates, the magnitude was much lower due to thestructural stabilization provided by the conjugated polymers thatmaintained CT globular structure. In a control experiment, it was alsoshown that free pSBAm-block-pNIPAm in solution had a small effect onstructural stabilization of native CT at pH 1 (FIG. 13b ). It wassurmised that structure stabilizing ions in the pSBAm block (ammoniumand sulfonate) of the free polymer slightly increased solution totalsalt concentration to stabilize CT tertiary structure. Whilestabilization was increased a small amount with free polymer insolution, polymer conjugation to the protein was superior for structuralstabilization due to the high local concentration of the polymer nearthe enzyme surface.

The stability of CT conjugates to pepsin degradation in acid was nextexplored. CT bioconjugates were stable to pepsin incubation at 37° C. in167 mM HCl (FIG. 13c ). Native CT lost all activity after only 75minutes, while CT conjugates maintained approximately 70% residualactivity after the same time period and still had 30% of initialactivity after 7 hours. For pepsin to cleave proteins throughhydrolysis, pepsin molecules must be able to access the specificallyfavored amino acid sequence on chymotrypsin. Due to the high densitypolymer shell around CT-pSBAm-block-pNIPAm conjugates of each molecularweight, access for pepsin was inhibited. In addition, due to the low pHconditions, unfolding occurred for native CT, which increased access forpepsin degradation.

Multi-stimuli responsive polymers represent a new class ofprotein-polymer conjugates that can respond to two stimuli throughseparate responses from each block. Temperature responsive polymerconjugation has previously been utilized for separation purposes (Chen,J. P.; Hoffman, A. S. Biomaterials 1990, 11, 631.), and the conjugatedescribed herein could be used for separations at both high and lowtemperature, which could be driven by the need to conserve energyrequired for high temperatures or prevent protein denaturation. Inaddition, protein-polymer conjugates with both UCST and LCST behaviorshow that other protein-polymer conjugates with separate blocks of pHand temperature responsive behavior can be synthesized as well. UCST andLCST are just two of the multitude of responsive properties that can beincorporated into block copolymers. A dual block conjugate with pH andtemperature sensitive behavior could be used to treat cancer byresponding to lower pH (Callahan, D. J.; Liu, W. E.; Li, X. H.; Dreher,M. R.; Hassouneh, W.; Kim, M.; Marszalek, P.; Chilkoti, A. Nano Lett.2012, 12, 2165) that is common in a tumor mass, as well as responding totargeted external heating at the tumor. (McDaniel, J. R.; Dewhirst, M.W.; Chilkoti, A. Int. J. Hyperthermia. 2013, 29, 501) The dual blocksystem lets scientists design the pH and temperature triggers morespecifically than could be done for a single polymer block with dualresponsiveness. Lastly, this approach shows that smart polymerfunctionality can be implemented with equal success on the inside oroutside block of a diblock polymer shell.

Conclusion

In Experimental Series IV, dual temperature responsiveCT-pSBAm-block-pNIPAm conjugates with different polymer chain lengthsand molecular weights were synthesized using a “grafting from” approachwith two successive ATRP reactions. Chain extension of the pNIPAm blockfrom homopolymer CT-pSBAm conjugate was utilized to grow the blockcopolymer from the surface of CT. From cloud point curves, it has beenshown that CT-35/39, CT-50/67, CT-90/100 conjugates each had both UCSTand LCST phase transition behavior. LCST behavior was due to the pNIPAmpolymer block and was not affected by chain length. The pSBAm polymerblock imparted UCST behavior on each of the conjugates, but the specifictransition temperature and behavior was dependent upon the chain lengthof attached polymers. Polymer conjugation to CT affected both enzymeturnover number (k_(cat)) and substrate affinity (K_(M)) values, butonly relative K_(M) values were dependent upon temperature. Relativeproductivity (k_(cat)/K_(M)) ratios were also dependent upontemperature, mostly due to observed effects of relative K_(M) values.Stability of CT-pSBAm-block-pNIPAm conjugates was dramatically higherthan native CT. Without being bound by theory, it is believed that thepolymers reduced protein unfolding and restricted access of proteasesthru steric hindrance for a variety of incubation conditions includingthermal, low pH, and protease degradation. The present invention hasbeen used to generates a protein that would be expected to survivepassage through the stomach. A variety of proteins may be modified withresponsive polymers that first protect enzymes from this environment andthen respond to stimuli as needed.

All publications, including associated Supplemental materials, arehereby incorporated by reference in their entirety as if each individualpublication was specifically and individually incorporated by reference.In case of conflict, the present application, including any definitionsherein, will control.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. While specificembodiments of the subject invention have been discussed, the abovespecification is illustrative and not restrictive. Many variations ofthe invention will become apparent to those skilled in the art uponreview of this specification. The full scope of the invention should bedetermined by reference to the claims, along with their full scope ofequivalents, and the specification, along with such variations. Suchequivalents are intended to be encompassed by the following claims.

The invention claimed is:
 1. A method comprising: in an aqueoussolution, immobilizing an active ester-functionalized amide-containingcontrolled radical polymerization CRP initiator comprised of thestructure

wherein X is a halogen or a chain transfer agent; R₁ is H or alkyl; R₂is an active ester moiety; and n is an integer from 1 to 6, on each of aplurality of amino binding sites on a protein surface to form aprotein-initiator conjugate; isolating the protein-initiator conjugate;mixing a first group of monomers having one or more desired propertieswith the protein-initiator conjugate; polymerizing the monomers from theprotein-initiator conjugate to grow a polymer under controlled radicalpolymerization conditions to form a protein-polymer conjugate; and,isolating the protein-polymer conjugate.
 2. The method recited in claim1 wherein immobilizing the initiator comprises mixing protein and theinitiator in a buffer at a pH of about 8 to 9 and stirring for a periodof time sufficient to allow the formation of covalent bonds between theinitiator and at least a majority of the amino binding sites.
 3. Themethod recited in claim 1 wherein isolating the protein-initiatorconjugate comprises removing unreacted and unattached compounds from thesolution.
 4. The method recited in claim 3 wherein removing unreactedand unattached compounds from the solution comprises passing thesolution through a dialysis membrane.
 5. The method recited in claim 4further comprises lyophilizing the protein-polymer conjugate.
 6. Themethod recited in claim 1 wherein the controlled radical polymerizationconditions comprise conditions for one of an atom radical polymerization(ATRP) procedure or a reversible-addition fragmentation chain transfer(RAFT) procedure.
 7. The method recited in claim 1 wherein thepolymerization is an ATRP procedure and the method further comprises:mixing the protein-initiator conjugate and monomers in a buffer andremoving oxygen from the mixture; separately adding a deoxygenatedligand to an aqueous copper catalyst solution; transferring thecopper-ligand catalyst solution to the protein-initiator conjugate andmonomer mixture; and stirring at 4-25° C. for a sufficient time to allowpolymerization.
 8. The method recited in claim 7 wherein removing oxygenfrom the protein-initiator conjugate and monomer mixture comprisesbubbling Ar or N₂ through the mixture.
 9. The method recited in claim 7wherein isolating the protein-polymer conjugate comprises passing themixture through a dialysis membrane and refrigerating for a period oftime sufficient to remove copper-ligand catalyst and unreacted monomer.10. The method recited in claim 7 wherein X in the initiator structureis one of Br, Cl, or F.
 11. The method recited in claim 7 wherein theinitiator comprises N-2-bromo-2-methylpropanoyl-β-alanineN′-oxysuccinimide ester.
 12. The method recited in claim 7 wherein theinitiator comprises N-2-chloro-propanoyl-β-alanine N′-oxysuccinimideester.
 13. The method recited in claim 1 wherein the active ester moietyis selected from the group consisting of N-oxysuccinimde ester,nitrophenyl ester, pentahalophenyl ester wherein the halogen is F or Cl,1-oxybenzotriazole ester, and 2-oxy-4,6-dimethyloxy-1,3,5-triazineester.
 14. The method recited in claim 1 wherein the polymerizationcomprises a RAFT procedure and the chain transfer agent comprises athiocarbonylthio agent.
 15. The method recited in claim 1 wherein theprotein comprises an enzyme and the initiator covalently binds to amajority of the binding sites on the surface of the enzyme.
 16. Themethod recited in claim 1 wherein the protein comprises an enzyme andthe initiator covalently binds to at least 85% of the binding sites onthe surface of the enzyme.
 17. The method recited in claim 1 wherein theenzyme is selected from the group consisting of chymotrypsin, lysozyme,β-Galactosidase, carbonic anhydrase, glucose oxidase, laccase, andacetylcholinesterase.
 18. The method recited in claim 1 wherein thepolymer comprises a stimuli responsive polymer that responds to at leastone stimulus.
 19. The method recited in claim 18 wherein the stimulus isone or both of pH and temperature.
 20. The method recited in claim 1wherein the protein-polymer conjugate comprises chymotrypsin modifiedthrough high density attachment of thermo-responsive polymers.
 21. Themethod recited in claim 1 wherein the protein-polymer conjugate formedfrom the controlled radical polymerization comprises aprotein-homopolymer conjugate of different polymer chain lengths and themethod further comprises: following the polymerization of the firstgroup of monomers from the protein-initiator conjugate, mixing a secondgroup of monomers having one or more desirable properties with theprotein-homopolymer conjugate under controlled radical polymerizationconditions to form a block copolymer.
 22. The method recited in claim 21wherein the block copolymer comprises a dual temperature responsiveenzyme-pSBAm-block-pNIPAm conjugate having different polymer chainlengths and molecular weights.
 23. The method recited in claim 1 whereinthe protein comprises an enzyme and the surface charge of the enzyme ismodified by growing cationic pQA from multiple sites on the surface ofenzyme.
 24. The method recited in claim 1 wherein the polymers grownfrom the plurality of surface amino sites of the protein core form ahigh density cationic polymer shell around the protein core.
 25. Themethod recited in claim 1 wherein the chain length of the polymers iscontrolled by adjusting the molar concentration of the first group ofmonomers added to the protein initiator conjugate to a desired amount.26. A macroinitiator comprising: a water soluble activeester-functionalized amide-containing controlled radical polymerizationinitiator comprised of the structure

wherein X is a halogen or a chain transfer agent; R₁ is H or alkyl; R₂is an active ester moiety; and n is an integer from 1 to 6, covalentlybound to each of a plurality of surface amino acid residues on aprotein.
 27. The macroinitiator recited in claim 26 wherein theinitiator further covalently binds to the N-terminus of the protein. 28.The macroinitiator recited in claim 26 wherein the initiator isN-2-bromo-2-methylpropanoyl-β-alanine N′-oxysuccinimide ester.
 29. Themacroinitiator recited in claim 26 wherein the initiator isN-2-chloro-propanoyl-β-alanine N′-oxysuccinimide ester.
 30. Themacroinitiator recited in claim 26 wherein the active ester moiety isselected from the group consisting of N-oxysuccinimde ester, nitrophenylester, pentahalophenyl ester, 1-oxybenzotriazole ester, and2-oxy-4,6-dimethyloxy-1,3,5-triazine ester.
 31. The macroinitiatorrecited in claim 26 wherein the initiator binds to at least 85% of thelysine residues on the protein.
 32. A composition comprising: a watersoluble active ester-functionalized amide-containing controlled radicalpolymerization initiator comprised of the structure

wherein X is a halogen or a chain transfer agent; R₁ is H or alkyl; R₂is an active ester moiety; and n is an integer from 1 to 6, covalentlybound to each of a plurality of surface amino acid residues on a proteinto form a densely modified protein-polymer conjugate with a density ofpolymer chains per unit surface area greater than one polymer chain per10 nm² of protein surface area.
 33. The composition recited in claim 32wherein the protein comprises an enzyme core having surface aminoresidues covalently bound at each of at least 85% of the surface aminoresidues to the initiator.
 34. The composition recited in claim 32wherein the densely modified protein-polymer conjugate comprises astimuli responsive polymer.
 35. The composition recited in claim 32wherein the densely modified protein-polymer conjugate comprises anenzyme selected from the group consisting of chymotrypsin, lysozyme,β-galactosidase, carbonic anhydrase, glucose oxidase, laccase, andacetylcholinesterase.
 36. The composition recited in claim 32 whereineach polymer chain comprises a block copolymer.
 37. The compositionrecited in claim 32 wherein the polymer chains form a shell around theprotein.
 38. The composition recited in claim 32 wherein the polymerchains comprise polypeptides with free amine groups.
 39. The compositionrecited in claim 32 wherein the polymer chains are formed from monomerspolymerizable by ATRP.